Process of producing fertile transgenic zea mays plants and progeny comprising a gene encoding phosphinothricin acetyl transferase

ABSTRACT

This invention relates to a reproducible system for the production of stable, genetically transformed maize cells, and to methods of selecting cells that have been transformed. One method of selection disclosed employs the Streptomyces bar gene introduced by microprojectile bombardment into embryogenic maize cells which were grown in suspension cultures, followed by exposure to the herbicide bialaphos. The methods of achieving stable transformation disclosed herein include tissue culture methods and media, methods for the bombardment of recipient cells with the desired transforming DNA, and methods of growing fertile plants from the transformed cells. This invention also relates to the transformed cells and seeds and to the fertile plants grown from the transformed cells and to their pollen.

This is a division of application Ser. No. 07/565,844 filed Aug. 9,1990, which was a continuation-in-part of Ser. No. 07/513,298, filedApr. 17, 1990, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to reproducible systems for geneticallytransforming monocotyledonous plants such as maize, to methods ofselecting stable genetic transformants from suspensions of transformedcells, and to methods of producing fertile plants from the transformedcells. Exemplary transformation methods include the use ofmicroprojectile bombardment to introduce nucleic acids into cells, andselectable and/or screenable marker systems, for example, genes whichconfer resistance (e.g., antibiotic, herbicide, etc.), or which containan otherwise phenotypically observable trait. In other aspects, theinvention relates to the production of stably transformed and fertilemonocot plants, gametes and offspring from the transgenic plants.

2. Description of the Related Art

Ever since the human species emerged from the hunting-gathering phase ofits existence, and entered an agricultural phase, a major goal of humaningenuity and invention has been to improve crop yield and to alter andimprove the characteristics of plants. In particular, man has sought toalter the characteristics of plants to make them more tasty and/ornutritious, to produce increased crop yield or render plants moreadaptable to specific environments.

Up until recent times, crop and plant improvements depended on selectivebreeding of plants with desirable characteristics. Initial breedingsuccess was probably accidental, resulting from observation of a plantwith desirable characteristics, and use of that plant to propagate thenext generation. However, because such plants had within themheterogenous genetic complements, it was unlikely that progeny identicalto the parent(s) with the desirable traits would emerge. Nonetheless,advances in controlled breeding have resulted from both increasingknowledge of the mechanisms operative in hereditary transmission, and byempirical observations of results of making various parental plantcrosses.

Recent advances in molecular biology have dramatically expanded man'sability to manipulate the germplasm of animals and plants. Genescontrolling specific phenotypes, for example specific polypeptides thatlend antibiotic or herbicide resistance, have been located withincertain germplasm and isolated from it. Even more important has been theability to take the genes which have been isolated from one organism andto introduce them into another organism. This transformation may beaccomplished even where the recipient organism is from a differentphylum, genus or species from that which donated the gene (heterologoustransformation).

Attempts have been made to genetically engineer desired traits intoplant genomes by introduction of exogenous genes using geneticengineering techniques. These techniques have been successfully appliedin some plant systems, principally in dicotyledonous species. The uptakeof new DNA by recipient plant cells has been accomplished by variousmeans, including Agrobacterium infection (32), polyethylene glycol(PEG)-mediated DNA uptake (25), electroporation of protoplasts (17) andmicroprojectile bombardment (23). Unfortunately, the introduction ofexogenous DNA into monocotyledonous species and subsequent regenerationof transformed plants has proven much more difficult than transformationand regeneration in dicotyledonous plants. Moreover, reports of methodsfor the transformation of monocotyledons such as maize, and subsequentproduction of fertile maize plants, have not been forthcoming.Consequently, success has not been achieved in this area and commercialimplementation of transformation by production of fertile transgenicplants has not been achieved. This failure has been particularlyunfortunate in the case of maize, where there is a particularly greatneed for methods for improving genetic characteristics.

Problems in the development of genetically transformed monocotyledonousspecies have arisen in a variety of general areas. For example, there isgenerally a lack of methods which allow one to introduce nucleic acidsinto cells and yet permit efficient cell culture and eventualregeneration of fertile plants. Only limited successes have been noted.In rice, for example, DNA transfer has only recently been reported usingprotoplast electroporation and subsequent regeneration of transgenicplants (41). Furthermore, in maize, transformation using protoplastelectroporation has also been reported (see, e.g., 17).

However, recovery of stably transformed plants has not beenreproducible. A particularly serious failure is that the few transgenicplants produced in the case of maize have not been fertile (38). Whileregeneration of fertile corn plants from protoplasts has been reported(37, 39), these reported methods have been limited to the use ofnon-transformed protoplasts. Moreover, regeneration of plants fromprotoplasts is a technique which carries its own set of significantdrawbacks. Even with vigorous attempts to achieve fertile, transformedmaize plants, reports of success in this regard have not beenforthcoming.

A transformation technique that circumvents the need to use protoplastsis microprojectile bombardment. Although transient expression of areporter gene was detected in bombarded tobacco pollen (47), stabletransformation by microprojectile bombardment of pollen has not beenreported for any plant species. Bombardment of soybean apical meristemswith DNA-coated gold particles resulted in chimeric plants containingtransgenic sectors. Progeny containing the introduced gene were obtainedat a low frequency (27). Bombardment of shoot meristems of immaturemaize embryos resulted in sectors of tissue expressing a visible marker,anthocyanin, the synthesis of which was triggered by the introduction ofa regulatory gene (46). An analysis of cell lineage patterns in maize(28) suggests that germline transformation of maize by such an approachmay be difficult.

A second major problem in achieving successful monocot transformationhas resulted from the lack of efficient marker gene systems which havebeen employed to identify stably transformed cells. Marker gene systemsare those which allow the selection of, and/or screening for, expressionproducts of DNA. For use as assays for transformed cells, the selectableor screenable products should be those from genetic constructsintroduced into the recipient cells. Hence, such marker genes can beused to identify stable transformants.

Of the more commonly used marker gene systems are gene systems whichconfer resistance to aminoglycosides such as kanamycin. While kanamycinresistance has been used successfully in both rice (51) and cornprotoplast systems (38), it remains a very difficult selective agent touse in monocots due to high endogenous resistance (19). Many monocotspecies, maize, in particular, possess high endogenous levels ofresistance to aminoglycosides. Consequently, this class of compoundscannot be used reproducibly to distinguish transformed fromnon-transformed tissue. New methods for reproducible selection of orscreening for transformed plant cells are therefore needed.

Accordingly, it is clear that improved methods and/or approaches to thegenetic transformation of monocotyledonous species would represent agreat advance in the art. Furthermore, it would be of particularsignificance to provide novel approaches to monocot transformation, suchas transformation of maize cells, which would allow for the productionof stably transformed, fertile corn plants and progeny into whichdesired exogenous genes have been introduced. Furthermore, theidentification of marker gene systems applicable to monocot systems suchas maize would provide a useful means for applying such techniquesgenerally. Thus, the development of these and other techniques for thepreparation of stable genetically transformed monocots such as maizecould potentially revolutionize approaches to monocot breeding.

SUMMARY OF THE INVENTION

The present invention addresses one or more of the foregoing or othershortcomings in the prior art by providing methods and compositions forthe preparation of stably transformed, monocotyledonous cells andsubsequent regeneration of fertile, transgenic plants and progeny,particularly maize.

It is therefore a particular object of the present invention to providetechniques that will allow one to prepare transgenic, fertile monocotssuch as maize which are preferably diploid and which have been stablytransformed through the introduction of a desired gene into its genome.

The present invention thus relates generally to methods for theproduction of transgenic plants. As used herein, the term transgenicplants is intended to refer to plants that have incorporated exogenousgenes or DNA sequences, including but not limited to genes or DNAsequences which are perhaps not normally present, genes not normallytranscribed and translated ("expressed") in a given cell type, or anyother genes of DNA sequences which one desires to introduce into thenon-transformed plant, such as genes which may normally be present inthe non-transformed plant but which one desires to have alteredexpression.

Exemplary genes which may be introduced include, for example, DNAsequences or genes from another species, or even genes or sequenceswhich originate with or are present in the same species, but areincorporated into recipient cells by genetic engineering methods ratherthan classical reproduction or breeding techniques. However, the termexogenous, is also intended to refer to genes which are not normallypresent in the cell being transformed, or perhaps simply not present inthe form, structure, etc., as found in the transforming DNA segment orgene, or genes which are normally present yet which one desires, e.g.,to have overexpressed. Thus, the term "exogenous" gene or DNA isintended to refer to any gene or DNA segment that is introduced into arecipient cell, regardless of whether a similar gene may already bepresent in such a cell.

An initial step in the production of fertile transgenic plants is theobtaining of a DNA composition, e.g., vectors, plasmids, linear DNAfragments, and the like, a component of which is to be delivered torecipient monocotyledonous cells. DNA segments for use in transformingsuch cells will, of course, generally comprise the gene or genes whichone desires to introduce into the cells. These genes can further includestructures such as promoters, enhancers, polylinkers, or even regulatorygenes as desired.

The construction of vectors which may be employed in practicing thepresent invention is generally within the skill of the art. (Seegenerally, refs 79, 80). Preferred constructs will generally include aplant promoter such as the CaMV 35S promoter (68), or others such asCaMV 19S (69), nos (70), Adh (71), sucrose synthase (72), thoseassociated with the R gene complex (96), or even tissue specificpromoters such as root cell promoters (73) and tissue specific enhancers(74). Constructs will also include the gene of interest along with a 3'end such as that from Tr7 or nos (75), or the like. Regulatory elementssuch as Adh intron 1 (76), sucrose synthase intron (77) or TMV omegaelement (78), may further be included where desired.

Certain elements may find utility when incorporated into genomes, evenwithout an associated expressible gene. For example, transposons such asAc, Ds or Mu are elements which can insert themselves into genes andcause unstable mutations. This instability apparently results fromsubsequent excision of the element from the mutant locus during plant orseed development. For a review covering the use of transposon elements,see references 56 and 57. These elements, particularly Ac, may beintroduced in order to inactivate (or activate) and thereby "tag" aparticular trait. Once tagged, the gene with this trait may be cloned,e.g., using the transposon sequence as a PCR primer together with PCRgene cloning techniques (58,59). Once identified, the entire gene(s) forthe particular trait, including control or regulatory regions wheredesired, may be isolated, cloned and manipulated as desired prior toreintroduction.

Another possible element which may be introduced is a matrix attachmentregion element (MAR), such as the chicken lysozyme A element (61), whichcan be positioned around an expressible gene of interest to effect anincrease in overall expression of the gene and diminish positiondependant effects upon incorporation into the plant genome (61, 62).

The generation and use of recipient cells is believed to be an importantaspect of the invention. As used herein, the term "recipient cell" isintended to refer to monocot cells that are receptive to transformationand subsequent regeneration into stably transformed, fertile monocotplants. The inventors thus propose that not all cells present in apopulation of cells subjected to transforming events will be "recipient"to successful transformation and regeneration. However, it is proposedthat through the application of the techniques disclosed herein, onewill be enabled to obtain populations which contain sufficient numbersof recipient cells to allow for successful stable transformation andregeneration.

Certain techniques are disclosed which may enrich for recipient cells.For example, it is believed that Type II callus development, followed bymanual selection and culture of friable, embryogenic tissue, results inan enrichment of recipient cells. Suspension culturing, particularlyusing the media disclosed in Table I herein, may also improve the ratioof recipient to non-recipient cells in any given population. In fact, itis proposed that media such as MS which has a high ammonia/nitrate ratiois counterproductive to the generation of recipient cells in that itpromotes loss of morphogenic capacity. N6 media on the other hand has asomewhat lower ammonia/nitrate ratio, and contains micronutrients such amolybdenum and manganese, and may promote the generation of recipientcells by maintaining cells in a proembryonic state capable of sustaineddivisions.

Manual selection of recipient cells, e.g., by selecting embryogeniccells from the surface of a Type II callus, is one means employed by theinventors in an attempt to enrich for recipient cells prior to culturing(whether cultured on solid media or in suspension). The preferred cellsmay be those located at the surface of a cell cluster, and may furtherbe identifiable by their lack of differentiation, their size and densecytoplasm. It is proposed that the preferred cells will generally bethose cells which are less differentiated, or not yet committed todifferentiation. Thus, one may wish to identify and select those cellswhich are cytoplasmically dense, evacuolated with a high nucleus tocytoplasm ratio (e.g., determined by cytological observations), smallsize (e.g., 10-20 μm), and capable of sustained divisions and proembryoformation.

It is proposed that other possible means of identifying such cells mightbe through the use of dyes such as Evan's blue which is excluded bycells with relatively non-permeable membranes (embryogenic cells tend tohave relatively non-permeable membranes). In contrast, Evan's blue tendsto be taken up by relatively differentiated cells such as rooty cellsand snake cells (so-called due to their snake-like appearance).

Other possible means of identifying recipient cells include the use ofisozyme markers of embryogenic cells, such as glutamate dehydrogenase,which can be detected by cytochemical stains (81). However, it isbelieved that the use of isozyme markers such as glutamate dehydrogenasemay lead to some degree of false positives from non-embryogenic cellssuch as rooty cells which nonetheless have a relatively high metabolicactivity.

Additionally, the inventors propose that cryopreservation may effect thedevelopment of, or perhaps select for, recipient cells. If such aselection occurs upon cryopreservation, it may be due to a selectionagainst highly vacuolated, non-embryogenic cells, which are perhapssomewhat selectively killed during cryopreservation.

The frequency of occurrence of cells receiving DNA is believed to below. Moreover, it is most likely that not all recipient cells receivingDNA segments will result in a transformed cell wherein the DNA is stablyintegrated into the plant genome and/or expressed. Some may show onlyinitial and transient gene expression. However, it is proposed thatcertain cells from virtually any monocot species may be stablytransformed through the application of the techniques disclosed herein.

The most preferred monocot will be the cereals such as maize. Withrespect to maize, the inventors propose that many of the techniques ofthe invention will be applicable to maize varieties in general, whetherinbred, elite inbred or hybrid varieties. It should be pointed out,though, that not all cell lines developed out of a particular variety orcross will necessarily show the same degree of stable transformability.For example, the present invention is exemplified through the use ofA188×B73 cell lines developed by standard techniques out of an A188×B73cross. The lines identified as SC716 and SC82 are examples of cellslines which were developed from an A188×B73 cross as describedhereinbelow. However, a number of other cell lines developed from thesame cross have not as yet proven to be stably transformable. Thus,stable transformability may not be immediately apparent with some lineseven from the same cross. (2 out of about 12 A188×B73 lines have provedto be stably transformable and yield fertile transgenic plants; about16% of the lines). Thus, where one desires to prepare transformants to aparticular cross or variety, it will generally be desirable to developseveral cell lines from the particular cross or variety (e.g., 8 to 10),and subject all of the lines so developed to the transformationprotocols hereof.

In order to improve the ability to identify transformants, one maydesire to employ a selectable or screenable marker gene as, or inaddition to, the expressible gene of interest. Marker genes code forphenotypes that allow cells which express the marker gene to bedistinguished from cells that do not have the marker. Such genes mayencode either a selectable or screenable marker, depending on whetherthe marker confers a trait which one can select for by chemical means,i.e., through the use of a selective agent (e.g., an herbicide,antibiotic, or the like), or whether it is simply a trait that one canidentify through observation or testing (e.g., the R-locus trait). Ofcourse, many examples of suitable marker genes are known to the art andcan be employed in the practice of the invention.

Possible selectable markers for use in connection with the presentinvention include but are not limited to a neo gene (82) which codes forkanamycin resistance and can be selected for using kanamycin, G418,etc.; a bar gene which codes for bialaphos resistance; a mutant EPSPsynthase gene (67) which encodes glyphosate resistance; a nitrilase genewhich confers resistance to bromoxynil (83); a mutant acetolactatesynthase gene (ALS) which confers imidazolinone or sulphonylurearesistance (60); or a methotrexate resistant DHFR gene (95). Where amutant EPSP synthase gene is employed, additional benefit may berealized through the incorporation of a suitable chloroplast transitpeptide (CTP; see ref. 94).

Exemplary screenable markers include a β-glucuronidase or uidA gene(GUS) which encodes an enzyme for which various chromogenic substratesare known; an R-locus gene, which encodes a product that regulates theproduction of anthocyanin pigments (red color) in plant tissues (59); aβ-lactamase gene (98), a gene which encodes an enzyme for which variouschromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a luciferase gene (84); a xylE gene (54) which encodes acatechol dioxygenase that can convert chromogenic catechols; anα-amylase gene (85); a tyrosinase gene (55) which encodes an enzymecapable of oxidizing tyrosine to DOPA and dopaquinone which in turncondenses to melanin; an α-galactosidase, which will turn a chromogenicα-galactose substrate.

Included within the terms "selectable or screenable marker genes" arealso genes which encode a secretable marker whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers which encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes which canbe detected catalytically. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA, small active enzymes detectable in extracellular solution (e.g.,α-amylase, β-lactamase, phosphinothricin transferase), or proteins whichare inserted or trapped in the cell wall (such as proteins which includea leader sequence such as that found in the expression unit of extensinor tobacco PR-S).

Of course, in light of this disclosure, numerous other possibleselectable and/or screenable marker genes will be apparent to those ofskill in the art. Therefore, the foregoing discussion is intended to beexemplary rather than exhaustive. Although the present disclosure isexemplified in detail through the use of the bar and/or GUS genes, theapplicable techniques for making and using any other screenable orselectable marker gene will be within the skill in the art in light ofthe present disclosure.

An illustrative embodiment of marker genes capable of being used insystems to select transformants is the bar gene from Streptomyces, suchas from the hygroscopicus species. The bar gene codes forphosphinothricin acetyl transferase (PAT) that inactivates the activeingredient in the herbicide bialaphos, phosphinothricin (PPT). PPTinhibits glutamine synthetase, (29, 47) causing rapid accumulation ofammonia and cell death. Success in use of this selective system in thecase of monocots was unexpected because of the major difficulties whichhave been encountered in transformation of cereals (36).

Where one desires to employ a bialaphos resistance gene in the practiceof the invention, the inventors have discovered that a particularlyuseful gene for this purpose is the bar gene obtainable from species ofStreptomyces (ATCC No. 21,705). The cloning of the bar gene has beendescribed (29, 45) as has the use of the bar gene in the context ofplants other than monocots (10, 11). However, in light of the techniquesdisclosed herein and the general recombinant techniques which are knownin the art, the introduction and use of any of the foregoing or othergenes is now possible.

The use of a gene from the maize R gene complex is proposed as aparticularly useful screenable marker. The R gene complex in maizeencodes a protein that acts to regulate the production of anthocyaninpigments in most seed and plant tissue. Maize strains can have one or asmany as four R alleles which combine to regulate pigmentation in adevelopmental and tissue specific manner. The present inventors haveapplied a gene from the R gene complex to maize transformation becauseit is viable, it is a naturally occurring product in maize, and it isvisually screenable without the need for additional assays. Thus, an Rgene introduced into such cells will cause the expression of a redpigment and, if stably incorporated, can be visually scored as a redsector. If a maize line is dominant for the enzymatic intermediates inthe anthocyanin biosynthetic pathway (C2, A1, A2, Bz1 and Bz2), butrecessive at the R locus, any cell from that line can be employed as arecipient for transformation. Exemplary lines include rg-Stadler inWisconsin 22 and TR112, a K55 derivative which is r-g, b, P1.

The inventors further propose that R gene regulatory regions may beemployed in chimeric constructs in order to provide mechanisms forcontrolling the expression of chimeric genes. More diversity ofphenotypic expression is known at the R locus than at any other locus(63). It is contemplated that regulatory regions obtained from regions5' to the structural R gene could be very valuable in directing theexpression of genes for, e.g., insect resistance, herbicide tolerance orother protein coding regions. For the purposes of the present invention,it is believed that any of the various R gene family members may besuccessfully employed (e.g., P, S, Lc, etc.). However, the mostpreferred will generally be Sn (particularly Sn:bo13). Sn is a dominantmember of the R gene complex and is functionally similar to the R and Bloci in that Sn controls the tissue specific deposition of anthocyaninpigments in certain seedling and plant cells. Therefore, its phenotypeis similar to R.

The choice of the particular DNA segments to be delivered to therecipient cells will often depend on the purpose of the transformation.One of the major purposes of transformation of crop plants is to addsome commercially desirable, agronomically important traits to theplant. Such traits include, but are not limited to, herbicideresistance, increased yields, insect and disease resistance, physicalappearance, food content and makeup, etc. For example, one may desire toincorporate one or more genes encoding herbicide resistance. The bar andglyphosate tolerant EPSP synthase genes are good examples. A potentialinsect resistance gene which can be introduced includes the Bacillusthuringiensis crystal toxin gene (86), which may provide resistance topests such as lepidopteran or coleopteran. Protease inhibitors may alsoprovide resistance (64). Moreover, the expression of juvenile hormoneesterase directed towards specific insect pests may also haveinsecticidal activity, or perhaps cause cessation of metamorphosis (65).

Genes encoding proteins characterized as having potential insecticidalactivity, such as the cowpea trypsin inhibitor (CpTI; 88) may find useas a rootworm deterrent; genes encoding avermectin (89,90) may proveparticularly useful as a corn rootworm deterent. Furthermore, genesencoding lectins may, additionally or alternatively, confer insecticideproperties (e.g., barley, wheat germ agglutinin, rice lectins, see ref.91), while others may confer antifungal properties (e.g., UDA (stingingnettle lectin), hevein, chitinase, see refs. 92, 93).

It is proposed that benefits may be realized in terms of increasedresistance to cold temperatures through the introduction of an"antifreeze" protein such as that of the Winder Flounder (87).

Ultimately, the most desirable "traits" for introduction into a monocotgenome may be homologous genes or gene families which encode a desiredtrait (e.g., increased yield per acre) and which are introduced underthe control of novel promoters or enhancers, etc., or perhaps evenhomologous or tissue specific (e.g., root specific) promoters or controlelements.

The invention thus contemplates that particular benefits may be realizedby the transformation of plant cells with any expressible gene, and isnot intended to be limited to the use of marker genes. As used herein,an "expressible gene" is any gene that is capable of being translatedinto a protein, expressed as a trait of interest, or the like, etc., andis not limited to selectable, screenable or non-selectable marker genes.The invention also contemplates that, where both an expressible genethat is not necessarily a marker gene is employed in combination with amarker gene, one may employ the separate genes on either the same ordifferent DNA segments for transformation. In the latter case, thedifferent vectors are delivered concurrently to recipient cells tomaximize cotransformation.

In certain embodiments, recipient cells are selected following growth inculture. Where employed, cultured cells will preferably be grown eitheron solid supports or in the form of liquid suspensions. In eitherinstance, nutrients may be provided to the cells in the form of media,and environmental conditions controlled. There are many types of tissueculture media comprising amino acids, salts, sugars, hormones andvitamins. Most of the media employed in the practice of the inventionwill have some similar components (see, e.g., Table 1 herein below), themedia differ in the composition and proportions of their ingredientsdepending on the particular application envisioned. For example, variouscell types usually grow in more than one type of media, but will exhibitdifferent growth rates and different morphologies, depending on thegrowth media. In some media, cells survive but do not divide.

Various types of media suitable for culture of plant cells have beenpreviously described. Examples of these media include, but are notlimited to the N6 medium described by Chu, et al. (5) and the MS media(30). In an exemplary embodiment for preparation of recipient cells, theinventors have modified these media (see, Table 1). A preferred hormonefor such purposes is dicamba or 2,4-D. However, other hormones may beemployed, including NAA, NAA +2,4-D or perhaps even picloram.Modifications of these and other basic media may facilitate growth ofrecipient cells at specific developmental stages.

An exemplary embodiment for culturing recipient corn cells in suspensioncultures includes using embryogenic cells in Type II callus, selectingfor small (10-30 μ) isodiametric, cytoplasmically dense cells, growingthe cells in suspension cultures with hormone containing media,subculturing into a progression of media to facilitate development ofshoots and roots, and finally, hardening the plant and readying itmetabolically for growth in soil. For use in transformation, suspensionculture cells may be cryopreserved and stored for periods of time,thawed, then used as recipient cells for transformation. It is proposedthat cryopreservation may serve to enrich for or promote the developmentof recipient cells. The inventors propose that there is a narrowtemporal window in which cultured cells retain their regenerativeability, thus, it is believed that they must be preserved at or beforethat temporal period if they are to be used for future transformationand regeneration.

An illustrative embodiment of cryopreservation methods comprises thesteps of slowly adding cryoprotectants to suspension cultures to give afinal concentration of 10% dimethyl sulfoxide, 10% polyethylene glycol(6000MW), 0.23 M proline, and 0.23 M glucose. The mixture is then cooledto -35° C at 0.5° C. per minute. After an isothermal period of 45minutes, samples are placed in liquid N₂. (Modification of methods ofWithers and King (49); and Finkle, et al.(15)). To reinitiate suspensioncultures from cryopreserved material, cells may be thawed rapidly andpipetted onto feeder plates similar to those described by Rhodes, et al.(38).

One embodiment of cultured plant cells that can serve as recipient cellsfor transforming with desired DNA segments, such as those which compriseexpressible genes, includes corn cells, more specifically, cells fromZea mays L. Somatic cells are of various types. Embryogenic cells areone example of somatic cells which may be induced to regenerate a plantthrough embryo formation. Non-embryogenic cells are those which willtypically not respond in such a fashion. An example of non-embryogeniccells are certain Black Mexican Sweet (BMS) corn cells, and these havebeen successfully transformed by microprojectile bombardment using theneogene followed by selection with the aminoglycoside, kanamycin (22).However, this BMS culture was not found to be regenerable, and generaluse of kanamycin may be hampered by endogenous resistance of maize (19).

Other recipient cell targets include, but are not limited to, meristemcells, Type I and II calli and gametic cells such as microspores andpollen. Pollen, as well as its precursor cells, microspores, may becapable of functioning as recipient cells for genetic transformation, oras vectors to carry foreign DNA for incorporation during fertilization.Direct pollen transformation would obviate the need for cell culture.Meristematic cells (i.e., plant cells capable of continual cell divisionand characterized by an undifferentiated cytological appearance,normally found at growing points or tissues in plants such as root tips,stem apices, lateral buds, etc.) may represent another type of recipientplant cell. Because of their undifferentiated growth and capacity fororgan differentiation and totipotency, a single transformed meristematiccell could be recovered as a whole transformed plant. In fact, it isproposed that embryogenic suspension cultures may be an in vitromeristematic cell system, retaining an ability for continued celldivision in an undifferentiated state, controlled by the mediaenvironment.

The development of embryogenic maize calli and suspension culturesuseful in the context of the present invention, e.g., as recipient cellsfor transformation, has been described in U.S. Ser. No. 06/877,033,filed Jun. 7, 1986, incorporated herein by reference.

There are many methods for introducing transforming DNA segments intocells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as by Agrobacterium infection ordirect delivery of DNA such as, for example, by PEG-mediatedtransformation, by electroporation or by acceleration of DNA coatedparticles, etc. Acceleration methods are generally preferred andinclude, for example, microprojectile bombardment and the like.Electroporation has been used to transform corn protoplasts (17).

An example of a method for delivering transforming DNA segments to plantcells is microprojectile bombardment. In this method, non-biologicalparticles may be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

A particular advantage of microprojectile bombardment, in addition to itbeing an effective means of reproducibly stably transforming monocots,is that neither the isolation of protoplasts (8) nor the susceptibilityof Agrobacterium infection is required. An illustrative embodiment of amethod for delivering DNA into maize cells by acceleration is aBiolistics Particle Delivery System, which can be used to propelparticles coated with DNA through a screen, such as a stainless steel orNytex screen, onto a filter surface covered with corn cells cultured insuspension. The screen disperses the tungsten-DNA particles so that theyare not delivered to the recipient cells in large aggregates. It isbelieved that without a screen intervening between the projectileapparatus and the cells to be bombarded, the projectiles aggregate andmay be too large for attaining a high frequency of transformation- Thismay be due to damage inflicted on the recipient cells by projectilesthat are too large.

For the bombardment, cells in suspension are preferably concentrated onfilters. Filters containing the cells to be bombarded are positioned atan appropriate distance below the macroprojectile stopping plate. Ifdesired, one or more screens are also positioned between the gun and thecells to be bombarded. Through the use of techniques set forth hereinone may obtain up to 1000 or more clusters of cells transientlyexpressing a marker gene ("foci") on the bombarded filter. The number ofcells in a focus which express the exogenous gene product 48 hourspost-bombardment often range from 1 to 10 and average 2 to 3.

After effecting delivery of exogenous DNA to recipient cells by any ofthe methods discussed above, a preferred step is to identify thetransformed cells for further culturing and plant regeneration. Thisstep may include assaying cultures directly for a screenable trait or byexposing the bombarded cultures to a selective agent or agents.

An example of a screenable marker trait is the red pigment producedunder the control of the R-locus in maize. This pigment may be detectedby culturing cells on a solid support containing nutrient media capableof supporting growth at this stage, incubating the cells at, e.g., 18°C. and greater than 180 μE m⁻² sec⁻¹, and selecting cells from colonies(visible aggregates of cells) that are pigmented. These cells may becultured further, either in suspension or on solid media.

An exemplary embodiment of methods for identifying transformed cellsinvolves exposing the bombarded cultures to a selective agent, such as ametabolic inhibitor, an antibiotic, herbicide or the like. Cells whichhave been transformed and have stably integrated a marker geneconferring resistance to the selective agent used, will grow and dividein culture. Sensitive cells will not be amenable to further culturing.

To use the bar-bialaphos selective system, bombarded cells on filtersare resuspended in nonselective liquid medium, cultured (e.g., for oneto two weeks) and transferred to filters overlaying solid mediumcontaining from 1-3 mg/l bialaphos. While ranges of 1-3 mg/l willtypically be preferred, it is proposed that ranges of 0.1-50 mg/l willfind utility in the practice of the invention. The type of filter foruse in bombardment is not believed to be particularly crucial, and cancomprise any solid, porous, inert support.

Cells that survive the exposure to the selective agent may be culturedin media that supports regeneration of plants. An example of suitablemedia is a modification of MS media (Table 1). Tissue is maintained on abasic media with hormones for about 2-4 weeks, then transferred to mediawith no hormones. After 2-4 weeks, shoot development will signal thetime to transfer to another media.

Regeneration typically requires a progression of media whose compositionhas been modified to provide the appropriate nutrients and hormonalsignals during sequential developmental stages from the transformedcallus to the more mature plant. Developing plantlets are transferred tosoil, and hardened, e.g., in an environmentally controlled chamber atabout 85% relative humidity, 600 ppm CO₂, and 250 microeinsteins m⁻²·s⁻¹ of light. Plants are preferably matured either in a growth chamberor greenhouse. Regeneration will typically take about 3-12 weeks. Duringregeneration, cells are grown on solid media in tissue culture vessels.An illustrative embodiment of such a vessel is a petri dish.Regenerating plants are preferably grown at about 19° to 28° C. Afterthe regenerating plants have reached the stage of shoot and rootdevelopment, they may be transferred to a greenhouse for further growthand testing.

To confirm the presence in the regenerating plants of traits deliveredto the recipient cells through the application of exogenous DNA, aloneor in conjunction with marker genes, assays for expression of said genesmay be performed, e.g., by testing parts of the regenerated plants.Exemplary parts which may be assayed are leaves. A typical transformantassay includes contacting regenerating plants or extracts of plants witha substrate that is acted upon by the transforming gene product. At thisstage of development, the plants will not be lethally affected by suchan assay. Removal of small portions of the plants does not cause theirdeath or interfere with further development.

In one study, R_(o) plants were regenerated from transformants of anA188×B73 suspension culture line (SC82) transformants, and these plantsexhibited a phenotype expected of the genotype of hybrid A188×B73 fromwhich the callus and culture were derived. The plants were similar inheight to seed-derived A188 plants (3-5 ft tall) but had B73 traits suchas anthocyanin accumulation in stalks and prop roots, and the presenceof upright leaves. It would also be expected that some traits in thetransformed plants would differ from their source, and indeed somevariation will likely occur.

In an exemplary embodiment, the proportion of regenerating plantsderived from transformed callus that successfully grew and reachedmaturity after transfer to the greenhouse was 97% (73 of 76). In oneexample, at least 50 viable progeny were recovered from R_(o) plants.R_(o) plants in the greenhouse were tested for fertility by backcrossingthe transformed plants with seed-derived plants by pollinating the R_(o)ears with pollen from seed derived B73 plants and this resulted inkernel development. Note, however, that kernels on transformed plantsmay require embryo rescue due to cessation of kernel development andpremature senescence of plants.

To rescue developing embryos, they are excised from surface-disinfectedkernels 10-20 days post-pollination and cultured. An embodiment of mediaused for culture at this stage comprises MS salts, 2% sucrose, and 5.5g/l agarose. In an illustrative embodiment of embryo rescue, largeembryos (defined as greater than 3 mm in length) are germinated directlyon an appropriate media. Embryos smaller than that were cultured for oneweek on media containing the above ingredients along with 10⁻⁵ Mabscisic acid and then transferred to hormone-free medium forgermination.

Progeny may be recovered from the transformed plants and tested forexpression of the exogenous expressible gene by localized application ofan appropriate substrate to plant parts such as leaves. In the case ofbar transformed plants, it was found that transformed parental plants(R_(o)) and their progeny (R₁) exhibited no bialaphos-related necrosisafter localized application of the herbicide Basta to leaves, if therewas functional PAT activity in the plants as assessed by an in vitroenzymatic assay. In one study, of 28 progeny (R₁) plants tested, 50%(N=14) had PAT activity. All PAT positive progeny tested contained bar,confirming that the presence of the enzyme and the resistance tobialaphos were associated with the transmission through the germline ofthe marker gene. The nonchimeric nature of the callus and the parentaltransformants (R_(o)) was suggested by germline transmission and theidentical Southern blot hybridization patterns and intensities of thetransforming DNA in callus, R_(o) plants and R₁ progeny that segregatedfor the transformed gene.

Genomic DNA may be isolated from callus cell lines and plants todetermine the presence of the exogenous gene through the use oftechniques well known to those skilled in the art. Note, that intactsequences will not always be present, presumably due to rearrangement ordeletion of sequences in the cell.

The inventors have been successful in producing fertile transgenicmonocot plants (maize) where others have failed. Aspects of the methodsof the present invention for producing the fertile, transgenic cornplants comprise, but are not limited to, development of suspensioncultures of recipient cells using media conducive to specific growthpatterns, choice of selective systems that permit efficient detection oftransformation; modifications of acceleration methods to introducegenetic vectors with exogenous DNA into cells; invention of methods toregenerate plants from transformed cells at a high frequency; and theproduction of fertile transgenic plants capable of surviving andreproducing.

DEFINITIONS

Callus--Proliferating mass of cells or tissue in vitro.

Type I--A compact, slow growing, heteromorphic callus(embryogenic/organogenic) which retains meristematic activity in regionsof organized tissue.

Type II--A friable, fast growing embryogenic callus composed ofaggregates of small isodiametric cells with dense cytoplasm. Oftencontains small embryoids attached to the underlying callus by asuspensor.

Embryogenic Callus--A type of callus capable of differentiating intosomatic embryos.

Germinal Cells (Gametes)--Cells of an organism which are capable oftransferring their genetic information to the next generation.

Genotype--The genetic complement of an organism. Heterologous DNA--DNAfrom a source different than that of the recipient cell.

Homologous DNA--DNA from the same source as that of the recipient cell.

Hybrid--Progeny resulting from a cross between parental lines.

Inbred Lines--Organisms that are genetically homogeneous (homozygous)resulting from many generations of self crossing.

In Vitro--In the laboratory.

In Vivo--In the living organism.

Monocot--Plants having a single cotyledon (the first leaf of the embryoof seed plants); examples include cereals such as maize, rice, wheat,oats and barley.

Non-Embryogenic Callus--A type of callus composed of undifferentiated,often highly vacuolated cells which are unable to be induced to formembryos.

Phenotype--Traits exhibited by an organism resulting from theinteraction of genotype and environment.

Protoplast--Plant cells exclusive of the cell walls.

Somatic Cells--Body cells of an organism, exclusive of germinal cells.

Transformation--Acquisition of new genetic coding sequences by theincorporation of added (exogenous) DNA.

Transgenic--Organisms (plants or animals) into which new DNA sequencesare integrated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of plasmids (vectors) used inbombardment experiments. The plasmids have been named (A) pDPG165 whichcontains bar, and (B) pDPG208 which contains uidA (the gene whichencodes β-glucuronidase (GUS)). Letters designate the locations ofvarious restriction sites, locations which may be cleaved by restrictionendonucleases, E, EcoRI; H, HindIII; B, BamHI; S, SmaI. A more detailedmap of pDPG165 is shown in (C), of pDPG208 in (D). In (E) is shown arestriction map of pAGUS1, also known as pDPG141, in which a the5'-noncoding and 5'-coding sequences were modified to incorporate theKozak consensus sequence and a HindIII restriction site. The sequenceshown is SEQ ID NO: 1. In (F) is shown a restriction map of pDPG237, aplasmid which contains Sn:bo13 cDNA, and in (G) is shown a map ofpDPG232, a plasmid which incorporates Rsn cDNA along with a 35S promoterand a Tr7 3' end.

FIG. 2. Appearance of cell colonies which emerge on selection plateswith bialaphos. Such colonies appear 6-7 weeks after bombardment. (A)SC82 bialaphos-resistant colony selected on 1 mg/l bialaphos. (B)Embryogenic SC82 bialaphos-resistant callus selected and maintained on 1mg/l bialaphos.

FIG. 3. Phosphinothricin acetyl transferase (PAT) activity inembryogenic SC82 callus transformants designated E1-E11 and anonselected control (EO). 25 μg of protein extract were loaded per lane.B13 is a BMS-bar transformant. BMS is Black Mexican Sweet corn.Activities of the different transformants varied approximately 10 foldbased on the intensities of the bands.

FIG. 4. Integration of the bar gene in bialaphos-resistant SC82 callusisolates E1-E11. DNA gel blot of genomic DNA (4 μg/digest) from El-Eliand a nonselected control (EO) digested with EcoRI and HindIII. Themolecular weights in kb are shown on the left and right. The blot washybridized with ³² P-labeled bar from pDPG165 (˜25×10⁶ Cerenkov cpm).Lanes designated 1 and 5 copies refer to the diploid genome and contain1.9 and 9.5 pg respectively of the 1.9 kb bar expression unit releasedfrom pDPG165 with EcoRI and HindIII.

FIG. 5. PAT Activity in Protein Extracts of R_(o) Plants. Extracts fromone plant derived from each of the four transformed regenerable calluslines from a suspension culture of A188×B73, SC82 (E10, E11, E2/E5, andE3/E4/E6) were tested for PAT activity (The designations E2/E5 andE3/E4/E6 represent transformed cell lines with identical DNA gel blothybridization patterns; the isolates were most likely separated duringthe culturing and selection process.) Protein extracts from anontransformed B73 plant and a Black Mexican Sweet (BMS) cell culturebar transformant were included as controls. Approximately 50 microgramsof total protein was used per reaction.

FIG. 6. DNA Gel Blot Analysis of Genomic DNA from Transformed Callus andCorresponding R_(o) Plants Probed with bar. Genomic DNA was digestedwith EcoRI and HindIII, which released the 1.9 kb bar expression unit(CaMV 35S promoter-bar-Tr7 3'-end) from pDPG165, the plasmid used formicroprojectile bombardment transformation of SC82 cells, and hybridizedto bar. The molecular weights in kb are shown on the left and right.Lanes designated E3/E4/E6, E11, E2/E5, and E10 contained 5 μg of eithercallus (C) or R_(o) plant DNA. The control lane contained DNA from anontransformed A188×B73 plant. The lane designated "1 copy" contained2.3 pg of the 1.9 kb EcoRI/HindIII fragment from pDPG165 representingone copy per diploid genome.

FIG. 7. PAT Activity and DNA Gel Blot Analysis of Segregating Progeny ofE2/E5 R_(o) Plants. (A) Analysis of PAT activity in ten progeny (lanesa-j) and a nontransformed control plant (lane k). Lanes designated a,b-h, i, and j contained protein extracts from progeny of separateparental R_(o) plants. The lane designated callus contained proteinextract from E2/E5 callus. Approximately 25 micrograms of total proteinwere used per reaction. (B) DNA gel blot analysis of genomic DNAisolated from the ten progeny analyzed in A. Genomic DNA (5 μg/lane) wasdigested with SmaI, which releases a 0.6 kb fragment containing bar frompDPG165, and hybridized with bar probe. The lane designated R_(o)contained DNA from the R_(o) parent of progeny a. The lane designated 1copy contained pDPG165 digested with SmaI to represent approximately 1copy of the 0.6 kb fragment per diploid genome (0.8 pg).

FIG. 8. Histochemical determination of GUS activity in bar-transformedSC82 callus line Y13. This bialaphos-resistant callus line, Y13, whichcontained intact GUS coding sequences was tested for GUS activity threemonths post-bombardment. In this figure, differential staining of thecallus was observed.

FIG. 9. Integration of exogenous genes in bialaphos-resistant SC716isolates R1-R21. (A) DNA gel blot of genomic DNA (6 μg/digest) fromtransformants isolated from suspension culture of A188×B73 (SC716),designated R1-R21, were digested with EcoRI and HindIII and hybridizedto ³² P-labeled bar probe (˜10×10⁶ Cerenkov cpm). Molecular weightmarkers in kb are shown on the left and right. Two copies of the barexpression unit per diploid genome is 5.7 pg of the 1.9 kb EcoRI/Hindfragment from pDPG165. (B) The blot from A was washed and hybridizedwith ³² P-labelled GUS probe (˜35×10⁶ Cerenkov cpm). Two copies of the2.1 kb GUS-containing EcoRI/HindIII fragment from pDPG208 is 6.3 pg.

FIG. 10. Functional Expression of Introduced Genes in Transformed R_(o)and R₁ Plants. (A) Basta^(R) resistance in transformed R_(o) plants. ABasta^(R) solution was applied to a large area (about 4×8 cm) in thecenter of leaves of nontransformed A188×B73 plant (left) and atransgenic R_(o) E3/E4/E6 plant (right). (B) Basta^(R) resistance intransformed R₁ plants. Basta^(R) was also applied to leaves of four R₁plants; two plants without bar (left) and two plants containing bar(right). The herbicide was applied to R₁ plants in 1 cm circles to fourlocations on each leaf, two on each side of the midrib. Photographs weretaken six days after application. (C) GUS activity in leaf tissue of atransgenic R_(o) plant. Histochemical determination of GUS activity inleaf tissue of a plant regenerated from cotransformed callus line Y13(right) and a nontransformed tissue culture derived plant (left). Bar=1cm. (D) Light micrograph of the leaf segment from a Y13 plant shown in(C), observed in surface view under bright field optics. GUS activitywas observed in many cell types throughout the leaf tissue(magnification=230X). (E) Light micrograph as in (D) of control leaf.

FIG. 11. Mature R_(o) Plant, Developing Kernels and Progeny. (A) Maturetransgenic R_(o) plant regenerated from an E2/E5 callus. (B) Progenyderived from an E2/E5 plant by embryo rescue; segregant bearing theresistance gene on the right, and lacking the gene on the left. (C)Using pollen from transformed R₁ plants to pollinate B73 ears, largenumbers of seed have been recovered. (D) A transformed ear from an R₁plant crossed with pollen from a non-transformed inbred plant.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the first time, fertile transgenic maize plants have been produced,opening the door to new vistas of crop improvement based on in vitrogenetic transformation. The inventors have succeeded where others havefailed by combining and modifying numerous steps in the overall processleading from somatic cell to transgenic plant. Although the methodsdisclosed herein are part of a unified process, for illustrativepurposes they may be subdivided into: culturing cells to be recipientsfor exogenous DNA; cryopreserving recipient cells; constructing vectorsto deliver the DNA to cells; delivering DNA to cells; assaying forsuccessful transformations; using selective agents if necessary toisolate stable transformants; regenerating plants from transformants;assaying those plants for gene expression and for identification of theexogenous DNA sequences; determining whether the transgenic plants arefertile; and producing offspring of the transgenic plants. The inventionalso relates to transformed maize cells, transgenic plants and pollenproduced by said plants.

A. Tissue Culture

Tissue culture requires media and controlled environments. "Media"refers to the numerous nutrient mixtures that are used to grow cells invitro, that is, outside of the intact living organism. The medium isusually a suspension of various categories of ingredients (salts, aminoacids, hormones, sugars, buffers) that are required for growth of mostcell types. However, each specific cell type requires a specific rangeof ingredient proportions for growth, and an even more specific range offormulas for optimum growth. Rate of cell growth will also vary amongcultures initiated with the array of media that permit growth of thatcell type.

Nutrient media is prepared as a liquid, but this may be solidified byadding the liquid to materials capable of providing a solid support.Agar is most commonly used for this purpose. Bactoagar and Gelgro arespecific types of solid support that are suitable for growth of plantcells in tissue culture.

Some cell types will grow and divide either in liquid suspension or onsolid media. As disclosed herein, maize cells will grow in suspension,but regeneration of plants requires transfer from liquid to solid mediaat some point in development. The type and extent of differentiation ofcells in culture will be affected not only by the type of media used andby the environment, for example, pH, but also by whether media is solidor liquid. Table 1 illustrates the composition of various media usefulfor creation of recipient cells and for plant regeneration.

B. Culturing Cells in Suspension to be Recipients for Transformation

It is believed by the inventors that the ability to prepare andcryopreserve suspension cultures of maize cells is an important aspectof the present invention, in that it provides a means for reproduciblyand successfully preparing cells for transformation. The studiesdescribed below set forth techniques which have been successfullyapplied by the inventors to generate transformable and regenerablesuspension cultures of maize cells. A variety of different types ofmedia have been developed by the inventors and employed in carrying outvarious aspects of the invention, including in particular, thedevelopment of suspension cultures. The following table, Table 1, setsforth the composition of the media preferred by the inventors forcarrying out these aspects of the invention.

                  TABLE 1                                                         ______________________________________                                        Illustrative Embodiments of Tissue Culture                                    Media Which are Used for Type II Callus                                       Development, Development of Suspension                                        Cultures and Regeneration of Plant Cells                                      (Specifically Maize Cells)                                                    Medium                                                                        Id.                 Su-  Optimal                                                                              Other                                         Number MS*    N6    crose                                                                              pH     Components**                                  ______________________________________                                         52    +      -     2%   6.0    0.25 mg thiamine                                                              1 mg 2,4-D                                                                    10.sup.-7 M ABA                                                               Bactoagar                                     101    +      -     3%   6.0    100 mg myo-inositol                                  v                        Bactoagar                                     142    +      -     6%   6.0    5 mg BAP                                             v                        0.186 mg NAA                                                                  0.175 mg IAA                                                                  0.403 mg 2-IP                                                                 200 mg myo-inositol                                                           Bactoagar                                     163    +      -     3%   6.0    3.3 mg dicamba                                       v                        100 mg myo-inositol                                                           Bactoagar                                     171    +      -     3%   6.0    0.25 mg 2,4-D                                        v                        10 mg BAP                                                                     100 mg myo-inositol                                                           Bactoagar                                     173    +      -     6%   6.0    5 mg BAP                                             v                        0.186 mg NAA                                                                  0.175 mg IAA                                                                  0.403 mg 2-IP                                                                 10.sup.-5 M ABA                                                               200 mg myo-inositol                                                           Bactoagar                                     177    +      -     3%   6.0    0.25 mg 2,4-D                                        v                        10 mg BAP                                                                     10.sup.-5 M ABA                                                               100 mg myo-inositol                                                           Bactoagar                                     201    -      +     2%   5.8    25 mM proline                                               v                 1 mg 2,4-D                                                                    100 mg casein hydrolysate                                                     Gelgro.sup.R                                  205    -      +     2%   5.8    25 mM proline                                               v                 0.5 mg 2,4-D                                                                  100 mg casein hydrolysate                                                     Gelgro.sup.R                                  227    -      +     2%   5.8    25 mM proline                                               v                 13.2 mg dicamba                                                               100 mg casein hydrolysate                                                     Gelgro.sup.R                                  401    +      -     3%   6.0    0.25 mg thiamine                                                              1 mg 2,4-D                                                                    2 mg NAA                                                                      200 mg casein hydrolysate                                                     500 mg K sulfate                                                              100 mg myo-inositol                                                           400 mg K phosphate                                                            (monobasic)                                   402    +      -     3%   6.0    0.25 mg thiamine                                                              25 mM proline                                                                 1 mg 2,4-D                                                                    200 mg casein hydrolysate                                                     500 mg K sulfate                                                              400 mg K phosphate                                                            (monobasic)                                                                   100 mg myo-inositol                           409    +      -     3%   6.0    0.25 mg thiamine                                                              25 mM proline                                                                 10 mg dicamba                                                                 200 mg casein hydrolysate                                                     500 mg K sulfate                                                              400 mg K phosphate                                                            (monobasic)                                                                   100 mg myo-inositol                           501    -      -     2%   5.7    Clark's***                                                                    Gelgro.sup.R                                  ______________________________________                                         *Basic MS medium described in reference 30. The medium described in ref.      30 is typically modified by decreasing the NH.sub.4 NO.sub.3 from 1.64 g/     to 1.55 g/l, and omitting the pyridoxine HCl, nicotinic acid, myoinositol     and glycine.                                                                  + = present; - = absent; v = vitamins                                         **NAA = Napthol Acetic Acid                                                   IAA = Indole Acetic Acid                                                      2IP = 2, isopentyl adenine                                                    2,4D = 2,4Dichlorophenoxyacetic Acid                                          BAP = 6benzyl aminopurine                                                     ABA = abscisic acid                                                           ***Basic medium described in reference 6                                 

Example 1: Initiation of the Suspension Culture GII(A188XB73)716(designated SC716) for Use in Transformation

This Example describes the development of a maize suspension culture,designated SC716, which was employed in various of the transformationstudies described hereinbelow. The Type II tissue used to initiate thecell suspension was derived from immature embryos of A188×B73 platedonto N6-based medium with 1 mg/ml 2,4-D (201; see Table 1). A Type IIcallus was initiated by visual selection of fast growing, friableembryogenic cells. The suspension was initiated within 6 months aftercallus initiation. Tissue chosen from the callus to initiate thesuspension consisted of very undifferentiated Type II callus, thecharacteristics of this undifferentiated tissue are the earliest stagesof embryo development along with the soft, friable, undifferentiatedtissue underlying it.

Approximately one gram of tissue was added to 20 mls of liquid medium.In this example, the liquid medium was medium 402 to which differentslow-release hormone capsule treatments were added (see Example 12below). These capsule treatments included 2,4-D, NAA, 2,4-D plus NAA,and 2 NAA capsules. One flask was initiated for each of the different402 media plus hormone combinations. Every 7 days each culture wassubcultured into fresh medium by transferring a small portion of thecellular suspension to a new flask. This involved swirling the originalflask to suspend the cells (which tend to settle to the bottom of theculture vessel), tilting the flask on its side and allowing the densercells and cell aggregates to settle slightly. One ml of packed cells wasthen drawn off from this pool of settled cells together with 4 mls ofconditioned medium. A sterile ten ml, wide tip, pipet was used for thistransfer (Falcon 7304). Any very large aggregates of cells which wouldnot pass easily through the pipet tip were excluded. If a hormonecapsule was present, it was also transferred to the new flask.

After approximately 7 weeks, the loose embryogenic cell aggregates beganto predominate and fragment in each of the cultures, reaching a statereferred to as "dispersed." The treatment which yielded the highestproportion of embryogenic clusters was the 402 medium plus a NAAcapsule. After the cultures became dispersed and were growing at a fastrate, doubling approximately every two to three days as determined byincrease in packed cell volume, a one ml packed cell inoculum from eachculture was transferred into 401 medium using a ten ml narrow tip pipet(Falcon 7551). These transfers were performed about every 3% days. Aninoculum from the 402 plus 2,4-D plus NAA capsules culture was also usedto initiate a culture in 409 medium (402 minus 2,4-D and plus 10mg/1dicamba) either with or without 1 ml coconut water (Gibco 670-8130AG).

The most dispersed cultures were cryopreserved after 2 weeks, 2 monthsor 5 months.

The culture grown on 409 with coconut water was brought out ofcryopreservation eight months later and thawed, cultured for two weekson solid 201 culture medium using BMS as a feeder layer (38) andtransferred to media 409 without coconut water. The culture wasmaintained by subculturing twice weekly, using 409 media, by the methoddescribed above.

Example 2: Initiation of the Suspension Culture (A188×B73)82 (designatedSC82) for Use in Transformation

This Example describes the development of another cell line employed invarious of the transformation studies set forth below, termed SC82. Inthe development of SC82, inoculum for suspension culture initiation wasvisually selected from a Type II callus that was derived from immatureembryos plated on a N6-based medium containing 13.2 mg/l dicamba (227)(Table 1). The suspension culture was initiated within 3 months ofinitiation of the Type II callus. Small amounts (50-100 mg) of callusdistinguishable by visual inspection because of its highly proembryonicmorphology, were isolated from more mature or organized structures andinoculated into a 50 ml flask containing 5 mls of filter-sterilizedconditioned medium from the various GII (A188×B73) 716 suspensioncultures (402 medium with four types of capsule treatments and 409medium).

After one week, this 5 ml culture was sieved through a 710 micron meshand used to inoculate 20 mls of corresponding fresh andfilter-sterilized conditioned medium from the established GII (A188×B73)716 cultures in 150 ml flasks. After one week or more of growth, two mlsof packed cells were subcultured to fresh media by the method describedabove. The suspension culture maintained on 409 by this method was thencryopreserved within 3 months. The original cell line, which wasmaintained on 409 (not a reinoculated cryopreserved culture) was used inexperiments 1 and 2 months later which resulted in stable transformationand selection (see Table 2 below). The cryopreserved culture was usedfor experiment 6 (see Table 2 below).

C. Slow Release Plant Hormone Capsules

Studies following the fate of radioactively labelled plant hormones(2,4-D and NAA) showed that within two days corn cells absorb most ofthe auxins present in suspension culture media. This problem of hormonedepletion can be overcome by spiking the cultures with a small amount ofauxin every other day. However, spiking cultures is very time consumingwhen done on a large scale and also increases the risk of contaminationas the culture vessels must be opened frequently. Slow release planthormone capsules were developed to overcome these problems. In summary,these capsules comprise a plant hormone, usually in a crystalline state,encapsulated in a silicone matrix surrounded by a silicone limitingmembrane. The rate of hormone release is controlled by the size of thediffusible area and the thickness of the membrane. They have theadvantages of 1) supplying hormones at an acceptable and predictablerate (e.g., 20-100 μg/20 ml culture media/day, 2) they are of aconvenient size (e.g., 0.5-1.5 cm in length) for use in liquid or solidculture medium, 3) they are very durable and easily sterilized byautoclaving, and 4) they can be stored dry until needed.

The present formulation involves the controlled release of a planthormone or selective agent for a plant tissue culture from an innermatrix containing crystals of the desired agent through an outerdiffusion limiting membrane. A preferred embodiment of the formulationis to mix 30% dry crystals of the desired agent with 70% (w/w) roomtemperature vulcanizing (RTV) silicone which is then injected intosilicone tubing having an appropriate diameter and wall thickness forthe desired release rate of the desired agent. (The preferred agents foremploying in connection with the slow release capsules are 2,4-D andNAA, and the preferred dimensions are 0.062"ID×0,125" OD).

The RTV silicone is then polymerized at room temperature or at a highertemperature to accelerate the vulcanization process. Followingvulcanization of the inner matrix, the tubing is cut to desired lengthsand the ends sealed with RTV silicone. The preferred lengths for use inconnection with the present invention are about 0.5 cm. After the endseals have polymerized, the resulting capsules can either be stored, asis, or autoclaved for 15 minutes on a fast exhaust cycle and storedindefinitely in a sterile form. Prior to use the capsules may beequilibrated to establish a stable diffusion gradient across themembrane, or used directly without equilibration.

Another formulation for a much lower release rate is to enclose crystalsof a desired substance suspended in a liquid such as water or siliconeoil in a relatively nonpermeable tubing such as Nylon-11. The releaserate from this reservoir can then be regulated by drilling various sizeholes in the tubing and gluing a silicone window over the hole withsilicone medical adhesive. Once again the capsules can be sterilized byautoclaving and stored dry until use.

An exemplary technique employed by the inventors for preparing slowrelease hormone capsules is as follows:

1. Two grams of Dow Corning MDX-4-4210 medical grade elastomer and 0.2grams of Dow Corning MDX-4-4210 curing agent were weighed into a 10 mlsyringe, the bottom of which was capped with a plastic cap.

2. Six-hundred mg of 2,4-D (or NAA), from which lumps have been removedby sieving through a 411μ stainless steel sieve, was added to the samesyringe and thoroughly mixed with the elastomer and curing agent.

3. The 10 ml syringe and its contents were then degassed for 1/2 hr in avacuum centrifuge to remove bubbles.

4. Dow Corning Silastic medical grade silicone tubing (0,062"ID×0,125"OD) of medium durometer (50 Shore A) was preswelled 10 to 30 minutes bysoaking in acetone.

5. The plastic cap was removed from the end of the 10 ml syringe and thedegassed silicone-2,4-D mixture was extruded into the preswollen tubingfrom which excess acetone had been removed by blowing a stream of airbriefly through it.

6. Both ends of the filled tubing were then clamped shut and the tubingheated at 50 degrees (the boiling point of acetone=56.5 degrees)overnight to accelerate the polymerization.

7. The tubing was then cut into 0.5 cm lengths.

8. The ends of the tubing sections were sealed with Dow Corning Type Amedical adhesive and allowed to dry for 24 hr.

9. The finished capsules are autoclaved dry for 15-20 min and stored dryuntil use.

10. Before use the capsules may be preequilibrated for 48 hr by shakingin 25 ml of sterile 1 to 10 mM KHCO₃, or added to cultures withoutequilibration.

D. Cryopreservation Methods

Cryopreservation is important not only because it allows one to maintainand preserve a cell culture for future use, but it also is believed bythe inventors that this may be a means for enriching for recipientcells.

Cell suspensions were cryopreserved using modifications of methodspreviously reported (15,49). The cryopreservation protocol comprisedadding a pre-cooled (0° C.) concentrated cryoprotectant mixture dropwiseover a period of one hour while stirring the cell suspension, which wasalso maintained at 0° C. during this period. The volume of addedcryoprotectant was equal to the initial volume of the cell suspension(1:1 addition), and the final concentration of cryoprotectant additiveswas 10% dimethyl sulfoxide, 10% polyethylene glycol (6000 MW), 0.23 Mproline and 0.23 M glucose. The mixture was allowed to equilibrate at 0°C. for 30 minutes, during which time the cell suspension/cryoprotectantmixture was divided into 1.5 ml aliquot (0.5 ml packed cell volume) in 2ml polyethylene cryo-vials. The tubes were cooled at 0.5° C./minute to-8° C. and held at this temperature for ice nucleation.

Once extracellular ice formation had been visually confirmed, the tubeswere cooled at 0.5° C./minute from -8° to -35° C. They were held at thistemperature for 45 minutes (to insure uniform freeze-induced dehydrationthroughout the cell clusters). At this point, the cells had lost themajority of their osmotic volume (i.e. there is little free water leftin the cells), and they could be safely plunged into liquid nitrogen forstorage. The paucity of free water remaining in the cells in conjunctionwith the rapid cooling rates from -35° to -196° C. prevented largeorganized ice crystals from forming in the cells. The cells are storedin liquid nitrogen, which effectively immobilizes the cells and slowsmetabolic processes to the point where long-term storage should not bedetrimental.

Thawing of the extracellular solution was accomplished by removing thecryo-tube from liquid nitrogen and swirling it in sterile 42° C. waterfor approximately 2 minutes. The tube was removed from the heatimmediately after the last ice crystals had melted to prevent heatingthe tissue. The cell suspension (still in the cryoprotectant mixture)was pipetted onto a filter, resting on a layer of agarose-immobilizedBMS cells (the feeder layer which provided a nurse effect duringrecovery). Dilution of the cryoprotectant occurred slowly as the solutesdiffused away through the filter and nutrients diffused upward to therecovering cells. Once subsequent growth of the thawed cells was noted,the growing tissue was transferred to fresh culture medium. The cellclusters were transferred back into liquid suspension medium as soon assufficient cell mass had been regained (usually within 1 to 2 weeks).After the culture was reestablished in liquid (within 1 to 2 additionalweeks), it was used for transformation experiments. When necessary,previously cryopreserved cultures may be frozen again for storage.

E. DNA Segments Comprising Exogenous Genes

As mentioned previously, there are several methods to construct the DNAsegments carrying DNA into a host cell that are well known to thoseskilled in the art. The general construct of the vectors used herein areplasmids comprising a promoter, other regulatory regions, structuralgenes, and a 3' end.

DNA segments encoding the bar gene were constructed into a plasmid,termed pDPG165, which was used to introduce the bialaphos resistancegene into recipient cells (see FIGS. 1A and FIG. 1C). The bar gene wascloned from Streptomyces hygroscopicus (53) and exists as a 559-bp Sma Ifragment in plasmid pIJ4101. The sequence of the coding region of thisgene is identical to that published (45). To create plasmid pDPG165, theSma I fragment from pIJ4104 was ligated into a pUC19-based vectorcontaining the Cauliflower Mosaic Virus (CaMV) 35S promoter (derivedfrom pBI221.1. provided by R. Jefferson, Plant Breeding Institute,Cambridge, England), a polylinker, and the transcript 7 (Tr7) 3' endfrom Agrobacterium tumefaciens (3' end provided by D. Stalker, Calgene,Inc., Davis, Calif.).

An additional vector encoding GUS, pDPG208, (FIGS. 1B and FIG. 1D) wasused in these experiments. It was constructed using a 2.1 kb BamHI/EcoRIfragment from pAGUS1 (provided by J. Skuzeski, University of Utah, SaltLake City, Utah) containing the coding sequence for GUS and the nos3'-end from Agrobacterium tumefaciens. In pAGUS1 the 5'-noncoding and5'-coding sequences for GUS were modified to incorporate the Kozakconsensus sequence (24) and to introduce a new HindIII restriction site6 bp into the coding region of the gene (see FIG. 1E). The 2.1 kbBamHI/EcoRI fragment from pAGUS1 was ligated into a 3.6 kb BamHI/EcoRIfragment of a pUC19-based vector pCEV1 (provided by Calgene, Inc.,Davis, Calif.). The 3.6 kb fragment from pCEV1 contains pUC19 and a 430bp 35S promoter from cauliflower mosaic virus adjacent to the firstintron from maize Adh1.

In terms of a member of the R gene complex for use in connection withthe present invention, the most preferred vectors contain the 35Spromoter from Cauliflower mosaic virus, the first intron from maizeAdh1, the Kozak consensus sequence, Sn:bo13 cDNA, and the transcript 73' end from Agrobacterium tumefaciens. One such vector prepared by theinventors is termed pDPG237. To prepare pDPG237 (see FIG. 1F), the cDNAclone of Sn:bo13 was obtained from S. Dellaporta (Yale University, USA).A genomic clone of Sn was isolated from genomic DNA of Sn:bo13 which hadbeen digested to completion with HindIII, ligated to lambda arms andpackaged in vitro. Plaques hybridizing to two regions of cloned Ralleles, R-nj and R-sc (97) were analyzed by restriction digest. A 2 kbSst-HincII fragment from the pSn7.0 was used to screen a cDNA libraryestablished in lambda from RNA of light-irradiated scutellar nodes ofSn:bo13. The sequence and a restriction map of the cDNA clone wasestablished.

The cDNA clone was inserted into the same plant expression vectordescribed for pDPG165, the bar expression vector (see above), andcontains the 35S Cauliflower mosaic virus promoter, a polylinker and thetranscript 7 3' end from Agrobacterium tumefaciens. This plasmid,pPDG232, was made by inserting the cDNA clone into the polylinkerregion; a restriction map of pDPG232 is shown in FIG. 1G. The preferredvector, pDPG237, was made by removing the cDNA clone and Tr7 3' end frompDPG232, with AvaI and EcoRI and ligating it with a BamHI/EcoRI fragmentfrom pDPG208. The ligation was done in the presence of a BamHI linker asfollows: (SEQ ID NO:2 and SEQ ID NO:3)

    ______________________________________                                        GATCCGTCGACCATGGCGCTTCAAGCTTC                                                 GCAGCTGGTACCGCGAAGTTCGAAGGGCT                                                 ______________________________________                                    

The final construct of pDPG237 contained a Cauliflower mosaic virus 35Spromoter, the first intron of Adh1, Kozak consensus sequence, the BamHIlinker, cDNA of Sn:Bo13, and the Tr7 3' end and is shown in FIG. 1F.

Additional vectors have been prepared using standard genetic engineeringtechniques. For example, a vector, designated pDPG128, has beenconstructed to include the neo coding sequence (neomycinphosphotransferase (APH(3')-II)). Plasmid pDPG128 contains the 35Spromoter from CaMV, the neomycin phosphotransferase gene from Tn5 (66)and the Tr7 terminator from Agrobacterium tumefaciens. Another vector,pDPG154, incorporates the crystal toxin gene and was also prepared bystandard techniques. Plasmid pDPG154 contains the 35S promoter, theentire coding region of the crystal toxin protein of Bacillusthuringiensis var. kurstaki HD 263, and the Tr7 promoter.

Various tandem vectors have also been prepared. For example, a bar/aroAtandem vector was constructed by ligating a blunt-ended 3.2 kb DNAfragment containing a mutant EPSP synthase aroA expression unit (93) toNdeI-cut pDPG165 that had been blunted and dephosphorylated (NdeIintroduces a unique restriction cut approximately 200 bp downstream ofthe Tr7 3'-end of the bar expression unit). Transformants having aroA inboth orientations relative to bar were identified.

F. Preferred Methods of Delivering DNA to Cells

A preferred DNA delivery system that does not require protoplastisolation or introduction of Agrobacterium DNA is microprojectilebombardment (8,23). There are several potential cellular targets formicroprojectile bombardment to produce fertile transgenic plants:pollen, microspores, meristems, and cultured embryogenic cells are but afew examples. Germline transformation in maize has not been previouslyreported by bombardment of any of these types.

One of the newly emerging techniques for the introduction of exogenousDNA constructs into plant cells involves the use of microprojectilebombardment. The details of this technique and its use to introduceexogenous DNA into various plant cells are discussed in Klein (1989;ref. 22), Wang, et al. (1988; ref. 50) and Christou, et al. (1988; ref.8). One method of determining the efficiency of DNA delivery into thecells via microprojectile bombardment employs detection of transientexpression of the enzyme β-glucuronidase (GUS) in bombarded cells. Forthis method, plant cells are bombarded with a DNA construct whichdirects the synthesis of the GUS enzyme.

Apparati are available which perform microprojectile bombardment. Acommercially available source is an apparatus made by Biolistics, Inc.(now DuPont), but other microprojectile or acceleration methods arewithin the scope of this invention. Of course, other "gene guns" may beused to introduce DNA into cells.

Several modifications of the microprojectile bombardment method weremade by the inventors. For example, stainless steel mesh screens wereintroduced below the stop plate of the bombardment apparatus, i.e.,between the gun and the cells. Furthermore, modifications to existingtechniques were developed by the inventors for precipitating DNA ontothe microprojectiles.

Example 3: Microprojectile Bombardment

For bombardment, friable, embryogenic Type-II callus (1) was initiatedfrom immature embryos essentially as set forth above in Examples 1 and2. The callus was initiated and maintained on N6 medium (5) containing 2mg/l glycine, 2.9 g/l L-proline, 100 mg/l casein hydrolysate, 13.2 mg/ldicamba or 1 mg/12,4-D, 20 g/l sucrose, pH 5.8, solidified with 2 g/lGelgro (ICN Biochemicals). Suspension cultures initiated from thesecallus cultures were used for bombardment.

In the case of SC82, suspension culture SC82 was initiated from Type-IIcallus maintained in culture for 3 months. SC82 cells (see Example 1)were grown in liquid medium for approximately 4 months prior tobombardment (see Table 2, experiments #1 and #2). SC82 cells were alsocryopreserved 5 months after suspension culture initiation, storedfrozen for 5 months, thawed and used for bombardment (experiment #6).

In the case of suspension culture SC716 (see Example 2), it wasinitiated from Type-II callus maintained 5 months in culture. SC716cells were cultured in liquid medium for 5 months, cryopreserved for 8months, thawed, and used two months later in bombardment experiments #4and #5. SC94 was initiated from 10 month old Type-II callus; andcultured in liquid medium for 5 months prior to bombardment (experiment#3).

Prior to bombardment, recently subcultured suspension culture cells weresieved through 1000 μm stainless steel mesh. From the fraction of cellclusters passing through the sieve, approximately 0.5 ml packed cellvolume (PCV) was pipetted onto 5 cm filters (Whatman #4) andvacuum-filtered in a Buchner funnel. The filters were transferred topetri dishes containing three 7 cm filters (Whatman #4) moistened with2.5 ml suspension culture medium.

The dish containing the filters with the immobilized cell suspensionswas positioned 6 cm below the lexan plate used to stop the nylonmacroprojectile. With respect to the DNA, when more than a singleplasmid was used, plasmid DNA was precipitated in an equimolar ratioonto tungsten particles (average diameter approximately 1.2 μm, GTESylvania) using a modification of the protocol described by Klein, etal. (1987, Ref. 23). In the modified procedure, tungsten was incubatedin ethanol at 65 degrees C. for 12 hours prior to being used forprecipitation. The precipitation mixture included 1.25 mg tungstenparticles, 25 μg plasmid DNA, 1.1 M CaCl₂ and 8.7 mM spermidine in atotal volume of 575 μl. After adding the components in the above order,the mixture was vortexed at 4° C. for 10 min, centrifuged (500 X G) for5 min and 550 μl of supernatant was decanted. From the remaining 25 μlof suspension, 1 μl aliquots were pipetted onto the macroprojectile forbombardment.

Each plate of suspension cells was bombarded twice at a vacuum of 28inches Hg. In bombarding the embryogenic suspensions of A188×B73 andA188×B84, 100 μm or 1000 μm stainless steel screens were placed about2.5 cm below the stop plate in order to increase the number of fociwhile decreasing their size and also to ameliorate injury to thebombarded tissue. After bombardment, the suspension cells and thesupporting filter were transferred onto solid medium or the cells werescraped from the filter and resuspended in liquid culture medium.

Cells from embryogenic suspension cultures of maize were bombarded withthe bar-containing plasmid pDPG165 alone or in combination with aplasmid encoding GUS, pDPG208 (FIG. 1). In experiments in which a GUSplasmid was included, two of the filters containing bombarded cells werehistochemically stained 48h post-bombardment. The total number of foci(clusters of cells) per filter transiently expressing GUS was at least1000. In two separate studies designed to quantitate transientlyexpressing cells (using an SC82 (A188×B73) suspension culture), the meannumber and standard deviation of GUS-staining foci per filter was 1472+/-211 and 2930 +/-(n=3 and 4, respectively). The number of cells inindividual foci that expressed GUS averaged 2-3 (range 1-10). Althoughhistochemical staining can be used to detect cells transformed with thegene encoding GUS, those cells will no longer grow and divide afterstaining. For detecting stable transformants and growing them further,e.g., into plants, selective systems compatible with viability arerequired.

G. Methods of Identifying Transformed Cells

It is believed that DNA is introduced into only a small percentage ofcells in any one experiment. In order to provide a more efficient systemfor identification of those cells receiving DNA and integrating it intotheir genomes, therefore, one may desire to employ a means for selectingthose cells that are stably transformed. One exemplary embodiment ofsuch a method is to introduce into the host cell, a marker gene whichconfers resistance to some agent, e.g. an antibiotic or herbicide. Thepotentially transformed cells are then exposed to the agent. In thepopulation of surviving cells are those cells wherein generally theresistance-conferring gene has been integrated and expressed atsufficient levels to survive. Cells may be tested further to confirmstable integration of the exogenous DNA. Using embryogenic suspensioncultures, stable transformants are recovered at a frequency ofapproximately 1 per 1000 transiently expressing foci. A specificembodiment of this procedure is shown in Example 5.

One of the difficulties in cereal transformation, e.g., corn, has beenthe lack of an effective selective agent for transformed cells, fromtotipotent cultures (36). Stable transformants were recovered frombombarded nonembryogenic Black Mexican Sweet (BMS) maize suspensionculture cells, using the neogene and selection with the aminoglycoside,kanamycin (22). This approach is limited because many monocots areinsensitive to high concentrations of aminoglycosides (12,19). The stageof cell growth, duration of exposure and concentration of theantibiotic, may be critical to the successful use of aminoglycosides asselective agents to identify transformants (26,51,52). In addition, useof the aminoglycosides, kanamycin or G418, to select stabletransformants from embryogenic maize cultures, in the inventors'experience, often results in the isolation of resistant calli that donot contain the neogene.

One herbicide which has been suggested in resistance studies is thebroad spectrum herbicide bialaphos. Bialaphos is a tripeptide antibioticproduced by Streptomyces hygroscopicus and is composed ofphosphinothricin (PPT), an analogue of L-glutamic acid, and twoL-alanine residues. Upon removal of the L-alanine residues byintracellular peptidases, the PPT is released and is a potent inhibitorof glutamine synthetase (GS), a pivotal enzyme involved in ammoniaassimilation and nitrogen metabolism (33). Inhibition of GS in plants byPPT causes the rapid accumulation of ammonia and death of the plantcells.

The organism producing bialaphos also synthesizes an enzymephosphinothricin acetyl transferase (PAT) which is encoded by the bargene. The use of the herbicide resistance gene encoding phosphinothricinacetyl transferase (PAT) is referred to in DE 3642 829 A wherein thegene is isolated from Streptomyces viridochromogenes. This enzymeacetylates the free amino group of PPT preventing auto-toxicity (45).The bar gene has been cloned (29,45) and expressed in transgenictobacco, tomato and potato plants (10) and Brassica (11). In previousreports, some transgenic plants which expressed the resistance gene werecompletely resistant to commercial PPT and bialaphos in greenhouses.

PCT Application No. WO 87/00141 refers to the use of a process forprotecting plant cells and plants against the action of glutaminesynthetase inhibitors. This application also refers to the use of suchof a process to develop herbicide resistance in determined plants. Thegene encoding resistance to the herbicide BASTA (Hoechstphosphinothricin) or Herbiace (Meiji Seika bialaphos) was said to beintroduced by Agrobacterium infection into tobacco (Nicotiana tabacum cvPetit Havan SR1), potato (Solanum tuberosum cv Benolima) and tomato(Lycopersicum esculentum) and conferred on plants resistance toapplication of herbicides.

An exemplary embodiment of vectors capable of delivering DNA to planthost cells is the plasmid, pDPG165. This plasmid is illustrated in FIG.1A and FIG. 1C. A very important component of this plasmid for purposesof genetic transformation is the bar gene which acts as a marker forselection of transformed cells.

Example 4: Selection of bar Transformants Using Bialaphos

The suspension culture (designated SC82) used in the initial experiments(see Example 3) was derived from embryogenic Type-II callus of A188×B73.Following bombardment (see Example 3), cells on filters were resuspendedin nonselective liquid medium, cultured for 1 to 2 weeks and transferredto filters overlaying solid medium containing 1 or 3 mg/l bialaphos. Thedegree of inhibition of tissue growth during selection was dependentupon the density of the cells on the filter and on the concentration ofbialaphos used. At the density plated (0.5 PCV/filter), the growth ofthe cells cultured on 1 mg/l bialaphos was only partially inhibited(˜30-50% of nonselected growth) and after 3 to 4 weeks much of thistissue was transferred as discrete clumps (˜5 mm in diameter) toidentical medium. 0n medium containing 3 mg/l bialaphos, the growth ofcells on the original selection filter was severely inhibited (˜10% ofnonselected growth) and selection was carried out without removing thetissue from the original filter.

Using either selection protocol (1 or 3 mg/l bialaphos), resistant cellcolonies emerged on the selection plates of SC82 bombarded with pDPG165approximately 6 to 7 weeks after bombardment (FIG. 2A).Bialaphos-resistant calli were maintained and expanded on selectionmedium. Much of this tissue was embryogenic (FIG. 2B). No colony growthoccurred on plates to which cells were added from suspension cultures onwhich no transforming attempts were made. These are controls whichconfirm the prediction that cells without the bar gene are not resistantto bialaphos.

Colonies on solid supports are visible groups of cells formed by growthand division of cells plated on such support. Colonies can be seen inFIG. 2A on a petri dish. In this figure, the cells capable of growth arethose that are resistant to the presence of the herbicide bialaphos,said resistance resulting from integration and expression of the bargene. Exposure of cells was to 1 mg/l bialaphos. FIG. 2B is amagnification showing the morphology of one bialaphos-resistant culturemaintained on selection media indicating that growth is embryogenic.

As a confirmation that the cells forming the colonies shown in FIG. 2Aand FIG. 2B had indeed incorporated the bar gene and were expressing it,bialaphos-resistant callus lines were analyzed for activity of the bargene product, phosphinothricin acetyl transferase (PAT), by thin-layerchromatography. Protein extracts from eleven callus lines (E1-11)isolated from SC82 bombardment experiments contained PAT activity asshown in FIG. 3 and activity levels varied approximately 10-fold amongthe isolates.

Still further and more direct confirmation of the presence of the bargene was obtained by analysis of the genomic DNA of potentialtransformants by DNA gel blots (FIG. 4). The sources of DNA which wereelectrophoresed through the gel were the bialaphos-resistant calluslines designated E1-E11 and a non-selected control, EO. FIG. 1A, FIG.1B, FIG. 1C and FIG. 1D indicates the cleavage sites of those enzymeswithin the bar gene plasmid.) After the DNA was electrophoresed throughthe gel and transferred to nylon membranes, the resulting blot washybridized with a ³² P-labeled bar gene sequence from the plasmidpDPG165. The radioactivity used per blot was approximately 25×10⁶Cerenkov cpm. The lane in FIG. 4 designated "1" and "5" copies contain1.9 and 9.5 pg respectively of the 1.9 kb bar expression unit releasedfrom the plasmid pDPG165 by application of the EcoRI and HindIIIenzymes; these amounts represent about 1 and 5 copies per diploidgenome.

Genomic DNA from all eleven bialaphos-resistant isolates containedbar-hybridizing sequences as shown in FIG. 4. The hybridization in allisolates to a fragment migrating slightly larger than 2 kb may be due tocontaminating pUC19 sequences contained in this bar probe preparation;no such hybridization occurred in subsequent experiments using the samegenomic DNA and a different preparation of the bar probe. Hybridizationto a 1.9 kb fragment in eight of the eleven isolates indicated thatthese isolates contained intact copies of the 1.9 kb bar expressionunit. The estimated copy numbers of the intact unit ranged from one ortwo (El, E7, E8, E10, E11) to approximately 20 (E3, E4, E6).Hybridization with the bar probe in isolates E2 and E5 occurred only toa single, higher molecular weight fragment (˜3 kb).

To establish that the PAT coding sequence was intact in isolates E2 andE5, genomic DNA was digested with SmaI, which releases a 559 bp fragmentcontaining the PAT structural gene (FIG. 1A), and subjected to DNA gelblot analysis using ³² P-labeled bar. This analysis confirmed thepresence of a single intact copy of bar. Expression of PAT in theseisolates may not be dependent on the 35S promoter or the Tr7 3' end. Thehybridization patterns of some of the isolates were identical (E2 andE5; E7 and E8; E3, E4, and E6); therefore, it is probable that someisolates did not arise from independent transformation events butrepresent transformants that were separated during selection.

Seven hybridization patterns were unique, likely representing sevenindependent single-cell transformation events. The patterns andintensities of hybridization for the seven transformants were unchangedduring four months in culture, providing evidence for the stability ofthe integrated sequences. The seven independent transformants werederived from two separate bombardment experiments. Four independenttransformants representing isolates E2/E5, E3/E4/E6, E1 and E7/E8, wererecovered from a total of four original filters from bombardmentexperiment #1 and the three additional independent transformants, E9,E10, and E11, were selected from tissue originating from six bombardedfilters in experiment #2. These data are summarized in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Summary of Maize Transformation Experiments                                                  # of    # with                                                                Independent                                                                           Intact                                                          # of  bar     bar   # with GUS                                                                           # with                                                                             Cointegration                                                                        Coexpression                  Exp.                                                                             Culture                                                                             Filters                                                                             Transformants                                                                         Expression                                                                          Coding GUS  Frequency                                                                            Frequency                     #  Bombarded                                                                           Bombarded                                                                           Recovered                                                                             Units Sequence                                                                             Activity                                                                           (%)    (%)                           __________________________________________________________________________    1  SC82  4     4       3     n.a.                                             2  SC82  6     3       2     n.a.                                             3  SC94  10    8       6     n.a.                                             4    SC716*                                                                            8     13      8     11     3    85     23                            5    SC716*                                                                            8     7       4      6     1    86     14                            6   SC82*                                                                              4     19      17    13     3    68     16                               Totals                                                                              40    54      40    30     7    77(30/39)                                                                            18(7/39)                      __________________________________________________________________________     *culture reinitiated from cryopreserved cells                                 n.a. not applicable; only pDPG165 DNA used or cotransformation analysis       not done                                                                 

Studies with other embryogenic suspension cultures produced similarresults. Using either an SC82 culture that was reinitiated fromcryopreserved cells (experiment #6) or an A188×B84 (SC94) suspensionculture (experiment #3), numerous independent transformants wererecovered (19 and 18 respectively; Table 2). All transformants containedthe bar gene and expressed PAT. The copy number of bar-hybridizingsequences and levels of PAT expression were comparable to the studiesdescribed above.

Example 5: Integration of the Bar Gene into Cell Lines Derived from theSC716 Suspension Culture

Bombardment studies and subsequent analyses were also performed on theA188×B73 suspension culture, termed SC716 (see Example 1). The resultanttransformed plant cells were analyzed for integration of bar genes. Tocarry out this analysis, genomic DNA was obtained from R1-R21 isolates;6 μg of DNA was digested with the restriction endonucleases EcoRI andHindIII, and DNA gel blot analysis was performed using the bar gene asprobe. In FIG. 9, molecular weights in kb are shown to the right andleft. The untransformed control is designated "RO," and the last columnto the right contains the equivalent of two copies of the bar geneexpression unit per diploid genome. For the DNA load used, two copiesthe bar expression unit per diploid genome is 5.7 pg of the 1.9 kbEcoRI/Hind fragment from the plasmid pDPG165. The DNA separated on thegel blot was hybridized to a ³² P-labeled bar probe. The label activityin the hybridization was approximately 10×10⁶ Cerenkov cpm. In A, thepresence of an intact bar expression unit is inferred from thehybridization of the bar probe to a 1.9 kb band in the gel.

Example 6: Assays for Integration and Expression of GUS

SC716 transformants discussed in Example 5, were further analyzed forintegration and expression of the gene encoding GUS. As determined byhistochemical assay, four of the SC716 transformants (R5, R7, R16, andR21) had detectable GUS activity 3 months post-bombardment. Expressionpatterns observed in the four coexpressing callus lines varied. Thenumber of cells with GUS activity within any given transformant sampledranged from ˜5% to ˜90% and, in addition, the level of GUS activitywithin those cells varied. The cointegration frequency was determined bywashing the genomic blot hybridized with bar (FIG. 9A) and probing with³² P-labeled GUS sequence as shown in FIG. 9B. EcoRI and HindIII, whichexcise the bar expression unit from pDPG165, also release from pDPG208 a2.1 kb fragment containing the GUS coding sequence and the nos 3' end(FIG. 1B).

Seventeen of the independent bar transformants contained sequences thathybridized to the GUS probe; three, R2, R14 and R19 did not.Transformants in which GUS activity was detected (R5, R7, R16 and R21)had intact copies of the 2.1 kb EcoRI/HindIII fragment containing theGUS structural gene (FIG. 9B). Transformants that contained largenumbers of fragments that hybridized to bar (R1, R5, R21) also containedcomparable number of fragments that hybridized to the gene encoding GUS(FIGS. 9A and 9B). This observation is consistent with those reportedusing independent plasmids in PEG-mediated transformation of A188 X BMSprotoplasts (26) and in studies conducted by the inventors involvingbombardment-mediated transformation of BMS suspension cells.

H. Co-Transformation

Co-transformation may be achieved using a vector containing the markerand another gene or genes of interest. Alternatively, different vectors,e.g., plasmids, may contain the different genes of interest, and theplasmids may be concurrently delivered to the recipient cells. Usingthis method, the assumption is made that a certain percentage of cellsin which the marker has been introduced, have also received the othergene(s) of interest. As can be seen in the following examples, not allcells selected by means of the marker, will express the other genes ofinterest which had been presented to the cells concurrently. Forinstance, in Example 7, successful cotransformation occurred in 17/20independent transformants (see Table 2), coexpression occurred in 4/20.In some transformants, there was variable expression among transformedcells.

Example 7: Co-Integration and Co-Expression of the Bar Gene and the GUSGene to Cell Lines Derived from the SC82 Suspension Culture

Of the bialaphos-resistant isolates selected from a reinitiation ofcryopreserved SC82 cells transformed with separate plasmids (asdescribed for SC716), nineteen independent transformants were selectedin this experiment (experiment #6, Table 2). The frequency ofcointegration and coexpression in those isolates was similar to thatdescribed for SC716 isolates (Table 2). The pattern of GUS staining inthese transformants varied in a manner similar to that described forcoexpressing SC716 transformants. A transformant, Y13, which containedintact GUS coding sequence, exhibited varying levels of GUS activity asshown in FIG. 8. This type of expression pattern has been describedpreviously in cotransformed BMS cells (22). Variable activity detectedin the cells from a single transformant may be attributed to unequalpenetration of the GUS substrate, or differential expression,methylation, or the absence of the gene in some cells.

These results show that both the bar gene and the GUS gene are presentin some of the cells bombarded with the two plasmids containing thesegenes. Co-transformation has occurred. In the cotransformation examplesdescribed herein and summarized in Table 2, cotransformation frequencyof the non-selected gene was 77%; coexpression frequency was 18%.

I. Regeneration of Plants From Transformed Cells

For use in agriculture, transformation of cells in vitro is only onestep toward commercial utilization of these new methods. Plants must beregenerated from the transformed cells, and the regenerated plants mustbe developed into full plants capable of growing crops in open fields.For this purpose, fertile corn plants are required. The inventiondisclosed herein is the first successful production of fertile maizeplants (e.g., see FIG. 11A) from transformed cells.

During suspension culture development, small cell aggregates (10-100cells) are formed, apparently from larger cell clusters, giving theculture a dispersed appearance. Upon plating these cells to solid media,somatic embryo development can be induced, and these embryos can bematured, germinated and grown into fertile seed-bearing plants. Thecharacteristics of embryogenicity, regenerability, and plant fertilityare gradually lost as a function of time in suspension culture.Cryopreservation of suspension cells arrests development of the cultureand prevents loss of these characteristics during the cryopreservationperiod.

One efficient regeneration system involves transfer of embryogeniccallus to MS (30) medium containing 0.25 mg/12,4-dichlorophenoxyaceticacid and 10.0 mg/16-benzyl-aminopurine. Tissue was maintained on thismedium for approximately 2 weeks and subsequently transferred to MSmedium without hormones (39). Shoots that developed after 2-4 weeks onhormone-free medium were transferred to MS medium containing 1% sucroseand solidified with 2 g/l Gelgro^(R) in Plant Con^(R) containers whererooting occurred.

Another successful regeneration scheme involved transfer of embryogeniccallus to N6 (5) medium containing 6% sucrose and no hormones (1) fortwo weeks followed by transfer to MS medium without hormones asdescribed above. Regeneration was performed at 25° C. under fluorescentlights (250 microeinsteins-m⁻² ·s⁻¹). After approximately 2 weeksdeveloping plantlets were transferred to soil, hardened off in a growthchamber (85% relative humidity, 600 ppm CO₂, 250 microeinsteins·m⁻²·s⁻¹) and grown to maturity either in a growth chamber or thegreenhouse.

Regeneration of plants from transformed cells requires careful attentionto details of tissue culture techniques. One of the major factors is thechoice of tissue culture media. There are many media which will supportgrowth of plant cells in suspension cultures, but some media give bettergrowth than others at different stages of development. Moreover,different cell lines respond to specific media in different ways. Afurther complication is that treatment of cells from callus initiationthrough transformation and ultimately to the greenhouse as plants,requires a multivariate approach. A progression consisting of variousmedia types, representing sequential use of different media, is neededto optimize the proportion of transformed plants that result from eachcell line. Table 3 illustrates sequential application of combinations oftissue culture media to cells at different stages of development.Successful progress is ascertained by the total number of plantsregenerated.

                                      TABLE 3                                     __________________________________________________________________________    Plants to Soil From Bombardment of SC716 (Expts 1, 2; Table 2).               REGENERATION MEDIA PROGRESSIONS                                                               227b                                                                             227b                                                                             227b                                                                             227b  227b                                                                             227b                                                                             227b  227b                                                                             #                                            227b                                                                             201b                                                                              52                                                                              163                                                                              205                                                                              227b                                                                             201b                                                                             205                                                                              163                                                                              227b                                                                             201b                                                                             PLANTS                                    227b                                                                             171                                                                              171                                                                              171                                                                              171                                                                              171                                                                              173                                                                              173                                                                              173                                                                              173                                                                              177                                                                              177                                                                              TO                              Cell Line 101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              SOIL                            __________________________________________________________________________    CONTROLS                                                                      A01C-11   X  4  X  X  X  X   2 X  X  X  X  X   6*                             A01C-01   X  7  X  X  X  X  27 X  X  X  X  X  34*                             TOTAL     X  11 X  X  X  X  29 X  X  X  X  X  40*                             TRANSFORMED                                                                   A01C-11   X  X  X  0  0  0  X  X  0  0  X  X  0                               A01C-12   X  2  X  0  0  0  X  X  0  0  X  X  2                               A01C-13   X  5  1  4  0  0   1  1 1  1  X  X  14*                             A01C-14   X  2  X  0  0  0  X  X  1  0  X  X   3*                             A01C-15   X  28 0  12 7  1  23 13 0  0  0  0  84*                             A01C-17   X  7  0  0  0  0  17  0 0  0  0  0  24                              A01C-18   X  12 0  0  X  0  21 10 0  X  2  0  45*                             A01C-19   X  0  X  X  0  X   0 X  X  0  X  0  0                               A01C-20   X  10 X  0  0  X   0 X  X  0  X  0  10*                             A01C-21   X  0  X  X  X  X   0 X  X  X  X  0  0                               A01C-24   2  4  0  0  0  0   6  5 0  0  0  0  17*                             A01C-25   X  9  X  X  0  0   1 X  0  0  X  X  10                              A01C-27   X  0  X  X  X  X  10 X  X  X  X  0  10*                             TOTAL     2  79 1  16 7  1  79 29 2  1  2  0  219*                            COMBINED                                                                      CONTROLS  X  11 X  X  X  X  29 X  X  X  X  X  40*                             TRANSFORMED                                                                             2  79 1  16 7  1  79 29 2  1  2  0  219*                            TOTAL     2  90 1  16 7  1  108                                                                              29 2  1  2  0  259*                            __________________________________________________________________________     X = Regeneration not attempted by this route.                                 * = More plants could have been taken to soil.                                201b = 201 with 1 mg/1 bialophos.                                             227b = 227 with 1 mg/1 bialophos.                                        

It can be seen that using the same group of media, cell lines will varyin their success rates (number of plants) (Table 3). There was alsovariation in overall success rate, line A01-15 yielding the greatestnumber of plants overall. (It should be noted, however, that becausetissue was limiting not all combinations of media were used on alllines, therefore, overall comparisons are limited.)

A preferred embodiment for use on cell lines in general, at leastinitially, is the combination shown in the second column under theregeneration media progression (media 227, 171, 101, 501). Media 227 isa good media for the selective part of the experiments, for example, touse for growth of callus in the presence of bialaphos. This mediacontains the hormone dicamba. NAA and 2,4-D are hormones in other media.In liquid media, these are usually encapsulated for controlled release(see Example 12 hereinbelow).

Thus, it can be seen from Table 1 that the various media are modified soas to make them particularly applicable to the development of thetransformed plant at the various stages of the transformation process.For example, subculture of cells in media 171 after applying theselective agent, yields very small embryos. Moreover, it is believedthat the presence of BAP in the media facilitates development of shoots.Myo-inositol is believed to be useful in cell wall synthesis. Shootelongation and root development proceeds after transfer to media 101.101 and 501 do not contain the hormones that are required for earlierstages of regeneration.

Transfer of regenerating plants is preferably completed in anagar-solidified media adapted from a nutrient solution developed byClark (1982; ref. 6), media 501. The composition of this mediafacilitates the hardening of the developing plants so that they can betransferred to the greenhouse for final growth as a plant. The saltconcentration of this media is significantly different from that of thethree media used in the earlier stages, forcing the plant to develop itsown metabolic pathways. These steps toward independent growth arerequired before plants can be transferred from tissue culture vessels(e.g. petri dishes, plant cans) to the greenhouse.

Approximately 50% of transformed callus lines derived from the initialSC82 and SC716 experiments were regenerable by the routes tested.Transgenic plants were regenerated from four of seven independent SC82transformants and ten of twenty independent SC716 transformants.

Regeneration of thirteen independently, transformed cell lines and twocontrol lines of SC716 was pursued. Regeneration was successful from tenof thirteen transformants. Although a total of 458 plantlets wereregenerated, due to time and space constraints only 219 transformedplants (representing approximately 48% of the total number ofregenerants) were transferred to a soilless mix (see below).Approximately 185 plants survived. Twelve regeneration protocols wereinvestigated and the number of plants regenerated from each route hasbeen quantified (Table 3). There appeared to be no significant advantageto maturing the tissues on 201, 52, 163, or 205 (see Table 1 for mediacodes) prior to transfer to medium 171 or 173. The majority of theplants were generated by subculturing embryogenic callus directly from227 to either 171 or 173. These plantlets developed roots withoutaddition of exogenous auxins, and plantlets were then transferred to asoilless mix, as was necessary for many of the transformants regeneratedfrom SC82.

The soilless mix employed comprised Pro Mix, Micromax, Osmocote 14-14-14and vermiculite. Pro Mix is a commercial product used to increasefertility and porosity as well as reduce the weight of the mixture. Thisis the bulk material in the mixture. Osmocote is another commercialproduct that is a slow release fertilizer with anitrogen-phosphorus-potassium ratio of 14:14:14. Micromax is anothercommercial fertilizer that contains all of the essential micronutrients.The ratio used to prepare the soilless mix was: 3 bales (3 ft³ each) ProMix; 10 gallons (vol.) vermiculite; 7 pounds Osmocote; 46 ml Micromax.The soilless mix may be supplemented with one or two applications ofsoluble Fe to reduce interveinal chlorosis during early seedling andplant growth.

Regeneration of transformed SC82 selected cell lines yielded 76 plantstransferred to the soilless mix, and 73 survived. The plants wereregenerated from six bialaphos-resistant isolates, representing four ofseven clonally independent transformants. Eighteen protocols were usedsuccessfully to regenerate the seventy six plants (Table 4). Differencesin morphology between cell lines deemed some protocols more suitablethan others for regeneration.

                                      TABLE 4                                     __________________________________________________________________________    EFFECTS OF PROGRESSION OF MEDIA ON THE NUMBER OF PLANTS REGENERATED           (SC82)*                                                                                                         227B     227B  227B                                                                             227B                                                                             227B                                227B                                                                             227B                                                                             227B                                                                             227B                                                                             227B                                                                             227B                                                                             227B                                                                             201B                                                                             227B  201B  201B                                                                             201B                                                                             201B                       227B                                                                             227B                                                                             227B                                                                             227A                                                                             227A                                                                             227A                                                                             171                                                                               52                                                                               52                                                                               52                                                                              171                                                                              201B                                                                             227B                                                                             205                                                                              227B                                                                             205                                                                                1                                                                               52                        142                                                                              173                                                                              171                                                                              205                                                                              209                                                                              173                                                                              173                                                                              173                                                                              173                                                                              171                                                                              173                                                                              173                                                                              178                                                                              171                                                                              177                                                                              177                                                                              178                                                                              171                    CELL                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101                                                                              101 # OF               LINE                                                                              501                                                                              501                                                                              501                                                                              501                                                                              501                                                                              501                                                                              501                                                                              501                                                                              501                                                                              501                                                                              501                                                                              501                                                                              501                                                                              501                                                                              501                                                                              501                                                                              501                                                                              501 PLANTS             __________________________________________________________________________    B3-14-4                                                                           1  X  14 X  X  X  1  1  X  2  X  X  5  X  5  X  X   X 29                  B3-14-9                                                                           X  X   1 1  X  4  1  X  X  X  X  X  X  1  X  1  X  X   9                  B3-14-7                                                                           X  X  X  X  X  X  X  X  X  X  6  2  X  X  X  X  X  1   9                  B3-14-6                                                                           X  X  X  X  1  X  X  X  X  X  X  X  X  X  X  X  X  X   1                  B3-14-3                                                                           X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X   0                  B3-14-2                                                                           X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X   0                  B3-14-1                                                                           X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X   0                  B3-14-5                                                                           X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X   0                  B3-13-5                                                                           X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X  X   0                  B3-13-2                                                                           X  1  13 X  X  X  3  2  2  X  X  X  X  X  1  X  X  X   22                 B3-13-1                                                                           X  3  X  1  X  X  X  X  1  X  X  X  X  X  X  X  1  X   6                  TO- 1  4  28 2  1  4  5  3  3  2  6  2  5  1  6  1  1  1   76                 TAL                                                                           __________________________________________________________________________     * = See table 1 for media codes.                                              X = This media progression was either attempted and unsuccessful or not       attempted.                                                                    227A = 227 with 10.sup.-7 M ABA.                                              227B = 227 with 1 mg/1 bialaphos.                                        

Prior to regeneration, the callus was transferred to either a) anN6-based medium containing either dicamba or 2,4-D or b) an MS-basedmedium containing 2,4-D. These steps allowed further embryoiddevelopment prior to maturation. Most of the maturation media containedhigh BAP levels (5-10mg/l) to enhance shoot development and causeproliferation. An MS-based medium with low 2,4-D (0.25 mg/l) and highBAP (10 mg/l), as described by Shillito, et al., (1989; Ref. 39) wasfound to be quite effective for regeneration.

Likewise, an MS-based medium containing 1 μm NAA, 1 μm IAA, 2 μm 2-IP,and 5 mg/l BAP (modified from 7) also promoted plant regeneration ofthese transformants. After plantlets recovered by any of theregenerative protocols had grown to five cm, they were transferred to anutrient solution described by Clark, (1982; Ref. 6) which wassolidified with Gelgro. Plantlets which were slow to develop roots weretreated with 3 μl droplets of 0.3% IBA at the base of the shoot tostimulate rooting. Plants with well developed root systems weretransferred to a soilless mix and grown in controlled environmentalchambers from 5-10 days, prior to transfer to the greenhouse.

J. Assays for Integration of Exogenous DNA and Expression of DNA inR_(o) R₁ Plants

Studies were undertaken to determine the expression of the transformedgene(s) in transgenic R_(o) and R₁ plants. Functional activity of PATwas assessed by localized application of a commercial herbicideformulation containing PPT to leaves of SC82 R_(o) and R₁ plants. Nonecrosis was observed on leaves of R_(o) plants containing either highlevels (E2/E5), or low levels (E3/E4) of PAT. Herbicide-treated E3/E4/E6and control leaves are shown in FIG. 10A. Herbicide was also applied toleaves of E2/E5 progeny segregating for bar. As demonstrated in FIG.10B, leaves of R₁ plants expressing bar exhibited no necrosis six daysafter application of the herbicide while R₁ plants without bar developednecrotic lesions. No necrosis was observed on transformed leaves up to30 days post-application.

Twenty-one R_(o) plants, representing each of the four regenerabletransformed SC82 callus lines, were also analyzed for expression of thebar gene product, PAT, by thin-layer chromatographic techniques. Proteinextracts from the leaves of the plants were tested. PAT activity of oneplant regenerated from each callus line is shown in FIG. 5.

All 21 plants tested contained PAT activity. Furthermore, activitylevels were comparable to levels in the callus lines from which theplants were regenerated. The nontransformed plant showed no PAT activity(no band is in the expected position for acetylated PPT in theautoradiograph from the PAT chromatogram). A band appears in the BMSlane that is not in lanes containing protein extracts from the plantleaves. This extra band was believed to be an artifact.

As another method of confirming that genes had been delivered to cellsand integrated, genomic (chromosomal) DNA was isolated from anontransformed plant, the four regenerable callus lines and from twoR_(o) plants derived from each callus line. FIG. 6 illustrates resultsof gel blot analysis of genomic DNA from the four transformed calli (C)and the R_(o) plants derived from them. The transformed callus and allplants regenerated from transformed callus contained sequences thathybridized to the bar probe, indicating the presence of DNA sequencesthat were complementary to bar. Furthermore, in all instances,hybridization patterns observed in plant DNA were identical in patternand intensity to the hybridization profiles of the corresponding callusDNA.

DNA from E3/E4/E6 callus and the desired R_(o) plants containedapproximately twenty intact copies of the 1.9 kb bar expression unit(Cauliflower Mosaic Virus 35S promoter-bar-Agrobacterium transcript 73'-end) as well as numerous other bar-hybridizing fragments. E11 callusand plant DNA contained 1-2 copies of the intact expression unit and 5-6additional non-intact hybridizing fragments. E10 callus and plantscontained 1-2 copies of the intact bar expression unit. E2/E5 DNAcontained a single fragment of approximately 3 kb that hybridized to theprobe. To confirm that the hybridizing sequence observed in all plantswere integrated into the chromosomal DNA, undigested genomic DNA fromone plant derived from each independent transformant was analyzed by DNAgel blot hybridization. Hybridization to bar was observed only in highmolecular weight DNA providing evidence for the integration of bar intothe maize genome.

Plants were regenerated from the coexpressing callus line, Y13, shown inFIG. 8. Plants regenerated from Y13 (experiment #6, Table 2) wereassayed for GUS activity and histochemically stained leaf tissue fromone plant is shown in FIG. 10C, FIG. 10D and FIG. 10E. Numerous celltypes including epidermal, guard, mesophyll and bundle sheath cellsstained positive for GUS activity. Staining intensity was greatest inthe vascular bundles. Although all leaf samples from the regeneratedplants tested (5/5) expressed the nonselected gene, some non-expressingleaf sectors were also observed. Leaf tissue extracts from three Y13 andthree control plants were also assayed for GUS activity by fluorometricanalysis (21). Activity detected in two opposing leaves from each ofthree Y13 plants tested was at least 100-fold higher than that incontrol leaves.

Example 8: General Methods for Assays

A method to detect the presence of phosphinothricin acetyl transferase(PAT) activity is to use thin layer chromatography.

An example of such detection is shown in FIG. 5 wherein various proteinextracts prepared from homogenates of potentially transformed cells, andfrom control cells that have neither been transformed nor exposed tobialaphos selection, are assayed by incubation with PPT and ¹⁴ C-AcetylCoenzyme A. 25 μg of protein extract were loaded per lane. The source inlanes E1-E11 were SC82 transformants; B13 is a BMS (Black Mexican Sweetcorn nonembryogenic) bar transformant. E0 is a nonselected,nontransformed control.

As can be seen at the position indicated by the arrow (the positionexpected for the mobility of ¹⁴ C-N-AcPPT), all lanes except thenontransformed control have activities with the appropriate mobility.Variation in activity among the transformants was approximately 10 fold,as demonstrated by the relative intensity of the bands. The results ofthis assay provide confirmation of the expression of the bar gene whichcodes for PAT. For analysis of PAT activity in plant tissue, 100-200 mgof leaf tissue was extracted in sintered glass homogenizers and assayedas described previously.

GUS activity was assessed histochemically as described using5-bromo-4-chloro-3-indolyl glucuronide (21) tissue was scored for bluecells 18-24 h after addition of substrate. Fluorometric analysis wasperformed as described by Jefferson (1987;Ref. 42) using 4-methylumbelliferyl glucuronide.

DNA gel blot analysis was performed as follows. Genomic DNA was isolatedusing a procedure modified from Shure, et al., (1983; Ref. 42).Approximately 1 gm callus tissue was ground to a fine powder in liquidN2 using a mortar and pestle. Powdered tissue was mixed thoroughly with4 ml extraction buffer (7.0 M urea, 0.35 M NaCl, 0.05 M Tris-HCl pH 8.0,0.01 M EDTA, 1% sarcosine). Tissue/buffer homogenate was extracted with4 ml phenol/chloroform. The aqueous phase was separated bycentrifugation, passed through Miracloth, and precipitated twice using1/10 volume of 4.4 M ammonium acetate, pH 5.2 and an equal volume ofisopropanol. The precipitate was washed with 70% ethanol and resuspendedin 200-500 μl TE (0.01 M Tris-HCl, 0.001 M EDTA, pH 8.0). Plant tissuemay also be employed for the isolation of DNA using the foregoingprocedure.

Genomic DNA was digested with a 3-fold excess of restriction enzymes,electrophoresed through 0.8% agarose (FMC), and transferred (43) toNytran (Schleicher and Schuell) using 10X SCP (20X SCP: 2 M NaCl, 0.6 Mdisodium phosphate, 0.02 M disodium EDTA). Filters were prehybridized at65° C. in 6X SCP, 10% dextran sulfate, 2% sarcosine, and 500 μg/mlheparin (4) for 15 min. Filters were hybridized overnight at 65° C. in6X SCP containing 100 μg/ml denatured salmon sperm DNA and ³² p-labeledprobe. The 0.6 kb SmaI fragment from pDPG165 and the 1.8 kb BamHI/EcoRIfragment from pCEV5 were used in random priming reactions (14);Boehringer-Mannheim) to generate labeled probes for detecting sequencesencoding PAT or GUS, respectively. Filters were washed in 2X SCP, 1% SDSat 65° C. for 30 min. and visualized by autoradiography using Kodak XAR5film. Prior to rehybridization with a second probe, the filters wereboiled for 10 min. in distilled H₂ O to remove the first probe and thenprehybridized as described above.

Example 9: Herbicide Application

The herbicide formulation used, Basta TX^(R) contains 200 g/lglufosinate, the ammonium salt of phosphinothricin. Young leaves werepainted with a 2% Basta solution (v/v) containing 0.1% (v/v) Tween-20.The prescribed application rate for this formulation is 0.5-1%.

In FIG. 10A, Basta^(R) solution was applied to a large area (about 4×8cm) in the center of leaves of a nontransformed A188×B73 plant (left)and a transgenic R_(o) E3/E4/E6 plant (right). In FIG. 10B, Basta wasalso applied to leaves of four R₁ plants; two plants without bar and twoplants containing bar. The herbicide was applied to R₁ plants in 1 cmcircles to four locations on each leaf, two on each side of the midrib.Photographs were taken six days after application.

K. Fertility of Transgenic Plants

To recover progeny the regenerated, genetically transformed maize plants(designated R_(o)), were backcrossed with pollen collected fromnontransformed plants derived from seeds, and progeny (designated R₁)that contained and expressed bar were recovered.

An important aspect of this invention is the production for the firsttime of fertile, genetically transformed maize plants (R_(o)) andprogeny (R₁). These were regenerated from embryogenic cells that weretransformed. R₁ plants are those resulting from backcrossing of R_(o)plants.

Pollination of transgenic R_(o) ears with non-transformed B73 pollenresulted in kernel development. In addition, kernels developed frompistillate flowers on male inflorescences that were pollinated withnon-transformed B73 pollen. Kernels on transformed R_(o) plants fromSC82 developed normally for approximately 10-14 days post-pollinationbut after this period the kernels ceased development and oftencollapsed. Most plants exhibited premature senescence at this time. Atotal of 153 kernels developed sporadically on numerous plants (seeTable 5): 8 of 37 E2/E5 plants, 2 of 22 E10 plants, and 3 of 6 E11plants. Viable progeny were recovered by embryo rescue from 11 E2/E5plants and one E10 plant.

                                      TABLE 5                                     __________________________________________________________________________    Regenerated Plants (R.sub.o) and progeny (R.sub.1)                                     # of                                                                          Independent                                                                           # of                                                                  bar     Regenerable                                                                          # of                                                                              #    # of R.sub.o                                                                        # of  # of                             Exp                                                                              Culture                                                                             Transformants                                                                         Transformed                                                                          R.sub.o                                                                           Reaching                                                                           Producing                                                                           Kernels                                                                             R.sub.1                          #  Bombarded                                                                           Recovered                                                                             Callus Lines                                                                         Plants                                                                            Maturity                                                                           Kernels                                                                             Recovered                                                                           Plants                           __________________________________________________________________________    1,2                                                                              SC82  7       4       76 73   23    153   40                               4,5                                                                               SC716                                                                              20      10     219 (35) (9)   (51)  (31)                             3  SC94  8        2.sup.a                                                                               11.sup.a                                                                         (0) (0)    (0)   (0)                             6  SC82  19       4.sup.a                                                                               23.sup.a                                                                         (0) (0)    (0)   (0)                             __________________________________________________________________________     .sup.a Regeneration in progress.                                              ()Experiment still in progress, data still being collected.              

SC716 R_(o) plants were also backcrossed with seed-derived B73 plants.To date, from the 35 mature SC716 R_(o) plants nine plants (representingfour independent callus lines) yielded 51 kernels, 31 of which producedvigorous R₁ seedlings (Table 5). Most kernels that developed on SC716plants did not require embryo rescue. Kernels often developed for 30-40days on the plant and some were germinated in soil. The remaining seedwas germinated on MS-based medium to monitor germination and transferredto soil after a few days. In addition to the improved kernel developmentobserved on SC716 R_(o) plants relative to SC82 R_(o) plants, pollendehisced from anthers of several SC716 plants and some of this pollengerminated in vitro (35). Transmission of the foreign gene has occurredboth through SC716 R₁ ears and using SC716 R₁ -derived pollen onnontransformed ears.

Pollen obtained from transformed R₁ plants has now been successfullyemployed to pollinate B73 ears and a large number of seeds have beenrecovered (see FIG. 11C). Moreover, a transformed ear from an R₁ plantcrossed with pollen from a non-transformed inbred plant is shown in FIG.11D. The fertility characteristics of the R₁ generation has beenconfirmed both from a standpoint of the pollen's ability to fertilizenon-transformed ears, and the ability of R₁ ears to be fertilized bypollen from non-transformed plants.

Example 10: Analysis of Progeny (R₁) of Transformed R_(o) Plants for PATand bar

A total of 40 progeny of E2/E5 R_(o) plants were analyzed for PATactivity, ten of which are shown in FIG. 7A. Of 36 progeny which wereassayed, 18 had PAT activity. Genomic DNA from the same ten progenyanalyzed for PAT activity was analyzed by DNA gel blot hybridization forthe presence of bar as shown in FIG. 7B. The six progeny tested thatexpressed PAT contained a single copy of bar identical in mobility tothat detected in callus and R_(o) plants; the four PAT-negative progenytested did not contain bar-hybridizing sequences. In one series ofassays, the presence of the bar gene product in 18 of 36 progenyindicates a 1:1 segregation of the single copy of bar found in E2/E5R_(o) plants and is consistent with inheritance of PAT expression as asingle dominant trait. A dominant pattern of inheritance would indicatethe presence in the plant of at least one copy of the gene coding forPAT. The single progeny recovered from an E10 R_(o) plant testedpositive for PAT activity.

It was determined that the methods disclosed in this invention resultedin transformed R_(o) and R₁ plants that produced functionally activePAT. This was determined by applying Basta (PPT) to the leaves of plantsand determining whether necrosis (tissue destruction) resulted from thisapplication. If functionally active PAT is produced by the plants, theleaf tissue is protected from necrosis. No necrosis was observed onR_(o) plants expressing high levels of PAT (E2/E5) or on plantsexpressing low levels (E3/E4/E6) (FIG. 10A).

Herbicide was also applied to leaves of R₁ progeny segregating for bar.In these studies, no necrosis was observed on R₁ plants containing andexpressing bar, however, necrosis was observed on those R₁ plantslacking the bar gene. This is shown in FIG. 10B.

Segregation of bar did not correlate with the variability in phenotypiccharacteristics of R₁ plants such as plant height and tassel morphology.In FIG. 9B, the plant on the right contains bar, the plant on the leftdoes not. In addition, most of the R₁ plants were more vigorous than theR_(o) plants.

Of the 23 R1 seedlings recovered to date from the SC716 transformants,ten of 16 had PAT activity. PAT activity was detected in four of tenprogeny from R_(o) plants representing callus line R18 and six of sixprogeny from R_(o) plants representing callus line R9.

L. Embryo Rescue

In cases where embryo rescue was required, developing embryos wereexcised from surface disinfected kernels 10-20 days post-pollination andcultured on medium containing MS salts, 2% sucrose and 5.5 g/l Seakemagarose. Large embryos (>3 mm) were germinated directly on the mediumdescribed above. Smaller embryos were cultured for approximately 1 weekon the above medium containing 10⁻⁵ M abscisic acid and transferred tohormone-free medium for germination. Several embryos became bacteriallycontaminated; these embryos were transferred to medium containing 300μg/ml cefoxitin. Developing plants were subsequently handled asdescribed for regeneration of R_(o) plants.

Example 11: Embryo Rescue

Viable progeny, recovered from seven SC82 E2/E5 plants and one SC82 E10plant, were sustained by embryo rescue. This method consisted ofexcising embryos from kernels that developed on R_(o) plants. Embryosranged in size from about 0.5 to 4 mm in length. Small embryos werecultured on maturation medium containing abscisic acid while largerembryos were cultured directly on germination medium. Two of theapproximately forty viable progeny thus far recovered from SC82 R_(o)plants are shown in FIG. 11B.

M. Phenotype of Transgenic Plants

Most of the R_(o) plants regenerated from SC82 transformants exhibitedan A188×B73 hybrid phenotype. Plants were similar in height to seedderived A188 plants (3-5 feet) but had B73 traits such as anthocyaninaccumulation in stalks and prop roots, and the presence of uprightleaves. Many plants, regardless of the callus line from which they wereregenerated, exhibited phenotypic abnormalities including leafsplitting, forked leaves, multiple ears per node, and coarse silk.Although many of the phenotypic characteristics were common to all R_(o)plants, some characteristics were unique to plants regenerated fromspecific callus lines. Such characteristics were exhibited regardless ofregeneration route and the time spent in culture during regeneration.

Nontransformed control plants were not regenerated from this cultureand, therefore, cannot be compared phenotypically. Pistillate flowersdeveloped on tassels of one E11 (1/6), several El0 (3/22) and almostone-third of the E2/E5 (12/37) plants with a range of three toapproximately twenty ovules per tassel. Primary and secondary earsdeveloped frequently on most E2/E5, E10, and E11 plants; a mature E2/E5plant is shown in FIG. 11A. Anthers rarely extruded from the tassels ofplants regenerated from SC82 transformants and the limited number ofanthers which were extruded did not dehisce pollen. Some phenotypiccharacteristics observed were unique to plants regenerated from aspecific callus line such as the lack of ears on E3/E4/E6 plants and a"grassy" phenotype (up to 21 lone narrow leaves) exhibited by all E11plants.

All SC82 plants senesced prematurely; leaf necrosis began approximatelytwo weeks after anthesis. The R_(o) plants regenerated from SC82transformed cell lines have tended to senesce prematurely; typicallybefore the developing kernels were mature. This has necessitated the useof embryo rescue to recover progeny (R₁ generation). Segregation of barin the R₁ generation does not correlate with the variability inphenotypic characteristics of R₁ plants such as plant height and tasselmorphology. In FIG. 11B, the plant on the right contains bar, the planton the left does not. In addition, most of the R₁ plants are morevigorous than the R_(o) plants. Transformed progeny (R1) have now alsobegun to yield kernels and R2 plantlets have been recovered.

Of 219 plants regenerated from 10 independent SC716 transformants,approximately 35 have reached maturity (Table 5). The SC716 plants didnot exhibit the phenotypic differences which characterized theindividual callus lines of SC82. These plants were more uniform andabnormalities less frequent. The phenotype of these plants closelyresembled that of control plants regenerated from a SC716 cryopreservedculture which was not bombarded. Plant height ranged from three to sixfeet with the majority of the plants between five and six feet. Mostmature plants produced large, multi-branched tassels and primary andsecondary ears. Pistillate flowers also developed on tassels of severalSC716 plants. Although anther extrusion occurred at approximately thesame low frequency as in the SC82 plants, a small amount of pollendehisced from some extruded anthers. For most of the SC716 plants thatreached maturity, senescence did not commence until at least 30 daysafter anthesis. The improved characteristics of SC716 plants over SC82plants indicate that differences between the suspension cultures may beresponsible.

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and herein be described in detail. It shouldbe understood, however, that it is not intended to limit the inventionto the particular forms disclosed, but on the contrary, the intention isto cover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the appended claims.

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAGGATCCGTC GACCATGGTAAGCTTAGCGGGCCCC36                                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GATCCGTCGACCATGG CGCTTCAAGCTTC29                                              (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GCAGCTGGTACCGCGAAGTT CGAAGGGCT29                                          

What is claimed is:
 1. A process for producing a fertile transgenic Zeamays plant comprising the steps of (i) establishing a regenerableculture from a Zea mays plant to be transformed, (ii) transforming saidculture by bombarding it with DNA-coated microprojectiles, wherein saidDNA comprises a selectable marker gene encoding for phosphinothricinacetyl transferase, (iii) identifying or selecting a transformed cellline and (iv) regenerating a fertile transgenic Zea mays planttherefrom, wherein said DNA is transmitted through a complete sexualcycle of said transgenic plant to its progeny, wherein said progenycomprises said selectable marker gene encoding phosphinothricin acetyltransferase, and wherein said gene is chromosomally integrated.
 2. Themethod of claim 1, wherein the gene encoding phosphinothricin acetyltransferase is the bar gene of Streptomyces hygroscopicus.
 3. The methodof claim 1, wherein the gene encoding phosphinothricin acetyltransferase is the bar gene of Streptomyces viridochromogenes.
 4. Themethod of claims 1, further comprising obtaining progeny of saidfertile, transgenic Zea mays plant, wherein said progeny is a fertile,transgenic Zea mays plant that comprises the gene encoding forphosphinothricin acetyl transferase.
 5. The method of claim 4, furthercomprising breeding said progeny with a non-transgenic maize plant, toprepare a fertile, transgenic Zea mays plant that comprises the geneencoding for phosphinothricin acetyl transferase.
 6. The method of claim4, further comprising breeding said progeny with a second transgenicmaize plant to prepare a fertile, transgenic Zea mays plant thatcomprises the gene encoding for phosphinothricin acetyl transferase. 7.The method of any one of claims 1 through 6, further comprisingpreparing seed from one or more fertile, transgenic Zea mays plants thatcomprises the gene encoding for phosphinothricin acetyl transferase,wherein said seed contains the gene encoding for phosphinothricin acetyltransferase.
 8. The method of claim 7, further comprising cultivatingsaid seed to prepare a fertile, transgenic Zea mays plant that comprisesthe gene encoding for phosphinothricin acetyl transferase.